seehsj south eastern europe health sciences journal southeastern european medical journal seemedj faculty of medicine osijek josip juraj strossmayer university of osijek j. huttlera 4 croatia – 31000 osijek seemedj@mefos.unios.hr seemedj.mefos.unios.hr we would like to thank our reviewers in 2022: ernst adeghate goran arbanas tatjana bacun ivana barac maja baretic lidija barić mirela baus loncar dino bekric nikola bijelić shubhada bopegamage tomislav bozek hrvoje brkic maja cigrovski berkovic erna davidovic-cvetko lorena duca ivana haršanji drenjančević marija jelic vukovic ivana jukić sandra karanovic miklos kellermayer aleksandar kibel tomislav kizivat sanja klobucar majanovic antonio kokot kristina kralik slavica kvolik ljiljana lukic ljiljana majnaric ana marusic martina mihalj dubravka mihaljevic dragan mirkov danijela nujic vlatka perisa marija renic markus ritter aleksandra rył oksana shevchuk vlatka sotosek mario stefanic southeastern european medical journal seemedj faculty of medicine osijek josip juraj strossmayer university of osijek j. huttlera 4 croatia – 31000 osijek seemedj@mefos.unios.hr seemedj.mefos.unios.hr ana stupin marko stupin krešimir šolić stana tokic serhii verevka andrea vukić dugac cahit yavuz oksana zayachkivska antje van der zee-neuen tatjana zemunik instead of an editorial: some statistics on first five-year period of publication of southeastern european medical journal (seemedj), from 2017-2022. ines drenjančević1, marija raguž1 1 faculty of medicine osijek, university josip juraj strossmayer osijek, j. huttlera 4, 31000 osijek, ines.drenjancevic@mefos.hr; maraguz@mefos.hr southeastern european medical journal (seemedj), has been published by faculty of medicine osijek, university josip juraj strossmayer osijek, since 2017. at the moment we have published 6 full volumens; this issue is the first of two, of the seventh volume. as a brand new scientific journal we have undergone all phases of development, necessity for recongnition in the scientific community as a desirable destination for quality manuscripts. one of the aims of seemedj is to provide support for young researchers who are at the beggining of their scientific and professional carriers, such as phd students. part of fullfiling that aim is that seemedj uses the diamond open access model. this means that there are no author processing fees and no fees to access the published papers. there are no article submission charges. the free access to full published manuscripts is available on our web page as well as in hrcak. in last five-year period, southeastern european medical journal (seemedj) has been covered by following databases and archives: indexing & abstracting services: doaj, google scholar (individual articles), oaji, publons, crossref, dimensions. journal directories: sherpa/romeo and digital preservation: hrčak following statistical data have been obtained from the seemedj archive. from 2017-2022, seemedj has published 105 scientific articles; 366 authors contributed to manuscripts, of which 6 % (22 authors) published 3 or more articles. majority of our authors come from croatia (figure 1. geographic origin of authors who have published in seemedj). however, we have authors from 9 countries and a number of different institutions. mailto:ines.drenjancevic@mefos.hr mailto:maraguz@mefos.hr https://hrcak.srce.hr/ https://oaji.net/journal-detail.html?number=10527 https://publons.com/journal/83357/southeastern-european-medical-journal/ http://www.crossref.org/ https://app.dimensions.ai/discover/publication?search_mode=content&order=date&and_facet_source_title=jour.1359233 http://www.sherpa.ac.uk/romeo/ https://hrcak.srce.hr/casopis/473 figure 1. the average number of views per article is shown in figure 2. the range is from 362 to 903 views per issue). at the moment seemedj has only 6 wos citation, 25 scopus and 160 google scholar (no duplicates). thus, our goals for future period are to increase visibility of the journal, to attract new authors from other countries and to increase quality of published work and subsequently increased citation and achieve indexation in other emintent indexation databases. figure 2. 903,1875 851,4444444 576,7142857 902,5 599,375 651,75 604,3333333 475,8 410,0588235 362,375 2017/1 2017/2 2018/1 2018/2 2019/1 2019/2 2020/1 2020/2 2021/1 2021/2 average number of views per article figure 3. 0 20 40 60 80 100 120 140 160 180 google scholar scopus wos citation count seehsj south eastern europe health sciences journal southeastern european medical journal seemedj faculty of medicine osijek josip juraj strossmayer university of osijek j. huttlera 4 croatia – 31000 osijek seemedj@mefos.unios.hr seemedj.mefos.unios.hr editorial seemedj 2022;6(1): 1-139 dear colleagues, hereby, the new, 11th issue of seemedj is presented to you. this issue brings 14 articles with the main topic of arterial hypertension. arterial hypertension is the leading cardiovascular disease, but also a risk factor for other cardiovascular and cerebrovascular disease and it is major public health problem due to its high prevalence. thus, this issue of seemedj is coedited with member of croatian academy of science, prof. bojan jelaković. topics covered in the present issue are hypertension management with sglt2 (e.g. invited review of biliććurčić et al), articles that assessed comorbidities and outcomes of arterial hypertension, particularly in relation to covid-19, still an actual topic (articles by gvozdanović et al and bukal et al). several reviews address other topic related to arterial hypertension, such as effects of anxiety, depression and antidepressant use on blood pressure by ivanušić-pejić et al, and the relationship of dehydroepiandrosterone sulfate and arterial hypertension (jug et al) and new markers for hypertension and cardiovascular diseases, non-coding parts mirnaa (kolobarić n and drenjančević i). adeleke presented a study on relationship between alcohol use and patterns of blood-pressure change due to examination stress among university academic stuff. in relation to the world and national campaigns for dietary salt intake reduction it is an intriguiging question if consumption of iodine is too high (elaborated by vasiljev et al). lack of physical activity is also an important contributing factor to bad hypertension outcomes. on the other hand, physical activity is necessary to preserve functional ability of elderly, which is studied by bilajac et al. kurup et al investigated transfusion transmissible infections among blood donors in the national blood transfusion service in guyana. mišković et al reviewed relationship of iron deficiency and recurrent aphtous stomatitis, while liović-milec et al presented their work on transient corneal edema after phacoemulsification, demonstrating that older patients, higher grade of no and amount of energy consumed during procedure are predictive factors for the severity of the corneal edema. during intramuscular vaccine injections, aspiration has always been performed, to ensure that the needle does not puncture one of the blood vessels. whether the lack of this simple procedure is related to side effects of vaccination southeastern european medical journal seemedj faculty of medicine osijek josip juraj strossmayer university of osijek j. huttlera 4 croatia – 31000 osijek seemedj@mefos.unios.hr seemedj.mefos.unios.hr (against covid-19, for example) is reviewed by kajan et al. finally, slivšek and co-authors introduce to us to the concept of deep ecology. we are happy to inform our potential authors that seemedj has been included to another citation base: oajiopen academic journal index and also pending to cabell’s database, thus widening our accessibility to readers. the art work at the cover page is a painting – a collage of osijek artist branimir kusik. i hope that readers will find relevant published articles for their work. on the behalf of the editorial board and my own, i warmly greet our readers and invite them to join us in the endeavor of publishing their own scientific work in seemedj. ines drenjančević, md, phd editor-in-chief southeastern european medical journal (seemedj) seehsj south eastern europe health sciences journal southeastern european medical journal seemedj faculty of medicine josip juraj strossmayer university of osijek cara hadrijana 10e croatia – 31000 osijek seemedj@mefos.unios.hr seemedj.mefos.unios.hr editorial dear colleagues, you have in your handsorbetter to sayat your sightthe first, inaugural issue of the southeastern european medical journal (seemedj) that is published by josip juraj strossmayer university of osijekfaculty of medicine osijek. there is no better time to start a new journal but in the anticipation of the 20th anniversary of the independent constitution of the faculty of medicine osijek as one of the higher educational institutions of osijek university. a wink in history in 1979, faculty of medicine in osijek started with 50 students in general medicine program, as a branch of faculty of medicine zagreb. in 1998, when it became independent, it had approximately 250 students; and in these 20 years, and particularly in the last 8 years it grew to almost 2200 students enrolled in many biomedical and health sciences, both undergraduate and graduate programs that we offer, including postgraduate doctoral study and having almost 200 teachers and faculties. such an enormous intellectual potential and diversity of programs that we present have put faculty of medicine osijek in the leading position in the region in education, but also in the scientific activities, including publications. faculty of medicine was one of the co-founders of the croatian medical journal, medicinski vjesnik and south east european health sciences journal. however, the need to have one’s own journal that will develop into the leading regional scientific journal offering an opportunity to publish in many fields of biomedicine and health sciences have outgrown these previous journals. upon the initiative of dean of the faculty of medicine osijek, aleksandar včev, seemedj was founded in january 2017 by the faculty council of the faculty of medicine osijek. seemedj is an international, peer reviewed, scientific and professional journal that presents original basic and applied research from all fields of medicine (general and clinical practice, and basic medical sciences). it seeks to promote the development and exchange of knowledge that is directly relevant to all spheres of nursing practice (ambulatory, community and family care, home, hospital, primary and secondary practice, and public health), as well as physiotherapy, sanitary and laboratory engineering, laboratory biotechnology, midwives and interdisciplinary studies. the editorial board is made of renowned scientists and medical professionals from osijek university and other croatian universities, neighborhood countries and countries as far as united stated of america. they all are friends, supporters and professors at the faculty of medicine osijek. their expertise grants the scientific merit and highest possible quality of the published papers, together with strict adherence to ethical principles in publishing. southeastern european medical journal seemedj faculty of medicine josip juraj strossmayer university of osijek cara hadrijana 10e croatia – 31000 osijek seemedj@mefos.unios.hr seemedj.mefos.unios.hr this seemedj vol 1, no 1, 2017, brings almost twenty papers, original research articles and reviews, authored by researchers from croatia, slovenia, hungary and ukraine. topics come from clinical medicine (i.e. internal medicine-cardiology, neurology, pediatrics, microbiology) to basic medical sciences (pharmacology, physiology), physiotherapy, nursing (community care), laboratory biotechnology and diagnosticsall in the scope of the journal. editorial board hopes that the readers will enjoy reading these papers and find them useful, mind widening and inspirational for future work. we certainly wish authors that their work published in seemedj achieve high impact on the biomedical scientific community and to live long and cite a lot. it is important to mention a art work at the cover page of the first issue. it is a painting by dragan melkus "polje poslije kiše (field after the rain)", 1911, from the gallery of fine arts in osijek (selected by ms valentina radoš, custos) dragan melkus (born in pozega) was our fellow citizen, renowned croatian writer and painter. selected painting is a presentation of our bellowed slavonian landscape. on a behalf of editorial board and my own, i warmly greet our readers and invite them to join us in the endeavor of publishing own scientific work in seemedj. sincerely, ines drenjančević, md, phd editor-in-chief southeastern european medical journal (seemedj) seemedj 2017, vol 1, no. 1 dirty croatian money: how big is the threat? 5 southeastern european medical journal, vol 1, 2017. dirty croatian money: how big is the threat?1 dino belić1, martina ilinić1, sven burian2, jasminka talapko1, domagoj drenjančević1,3 1 faculty of medicine, josip juraj strossmayer university of osijek, osijek, croatia 2 department of transfusion medicine, general hospital pula, pula, croatia 3 clinical institute of transfusion medicine, university hospital center osijek, osijek, croatia corresponding author: domagoj drenjančević, md, phd domagoj@mefos.hr received: march 14, 2017; revised version accepted: march 29, 2017; published: april 24. 2017 keywords: bacterial contamination, normal bacterial flora, banknotes, coins abstract aim: the objective of this study was to determine the extent of bacterial contamination, expressed as colony forming units (cfu), on individual banknotes and coins of croatian kuna (hrk). the purpose of the study was to define if the fear of money-transmitted diseases is founded. methods: one-hundred twenty pieces of banknotes and coins were collected for the experiment, 10 bills of 10, 20, 50, 100, 200 and 500 hrk and 10 coins of 10, 20 and 50 croatian lipa and 1, 2 and 5 hrk. at the department of microbiology and parasitology, faculty of medicine, university of osijek, swabs were taken from money, moistened in saline, planted on blood agar and incubated for 24 hours under ambient conditions at 37 °c. after growing the bacteria, cfu were counted and replanted for further identification, which was performed in accordance with the microbiological professional standards. results: in total, 739 bacterial cfu were grown and six bacterial species have been identified: staphylococcus epidermidis, staphylococcus saprophyticus, streptococcus viridans, bacillus sp., klebsiella sp., neisseria sp. almost 30% of the money was bacteriologically clean. there were no statistical differences between the prevalence of bacterial contamination of banknotes and coins. the most common bacteria isolated was s. epidermidis (86.33%) with statistical significance both on banknotes and coins (p<0.0001). conclusion: the identified bacterial species are mostly part of the normal human flora. pathogenic, and potentially pathogenic bacterial species were not found on croatian banknotes and coins in a respect for one colony of klebsiella sp. (belić d, ilinić m, burian s, talapko j, drenjančević, d. dirty croatian money: how big is the threat? seemedj 2017;1(1);5-10) seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 6 southeastern european medical journal, vol 1, 2017. introduction the conventional wisdom is that the money is dirty and contaminated with microorganisms that cause diseases. generally, money keeps circulating from person to person, from device to device and probably is the most frequently exchanged article between human beings. the microorganisms present on money are part of the normal human flora or pathogens such as bacteria, viruses and fungi. microbial contaminants may be transmitted directly, through hand-to-hand contact, or indirectly, via food or other inanimate objects. cause for special concern is the possibility of transmission of nosocomial pathogens that represent one of the most important global threats of our time, such as the spread of multi-resistant bacteria. for example, methicillin-resistant staphylococcus aureus (mrsa) is one of the most resistant nosocomial pathogens. this pathogen can survive on banknotes and coins (1). medical staff and patients who do not observe prescribed protection measures (hand washing and disinfection) before and after a direct contact with an infected person are the most common carriers of mrsa. there is no person that did not suffer from diarrhea at least once in their life. diarrhea is mainly caused by enteric pathogens such as enterotoxigenic e. coli, vibrio cholerae, salmonella. it has been proven in welldesigned experimental research that bacteria mentioned above can survive on money surface (1, 2). although this is an important issue, currently there is limited literature available. in this research, we are trying to answer some important questions regarding the hygienic safety of croatian money and its role in transmission of infectious diseases and to clarify some urban legends and fears regarding the impurity of money. indeed, what kind of money is dirty? methods we collected 120 pieces of banknotes and coins for the experiment, 10 pieces (paper money) of 10, 20, 50, 100, 200 and 500 croatian kuna (hrk) and 10 coins of 10, 20 and 50 croatian lipa and 1, 2 and 5 hrk. these different denominations of hrk were circulating between people and were collected during everyday activities: in various shops and a bank. in the laboratory of the department of microbiology and parasitology, faculty of medicine, university of osijek, plain sterile swabs (copan italia s.p.a, brescia, italy) were taken for complete notes and coins (on both sides). swabs were moistened in saline, plated on blood agar plate (bd, sparks, usa) and incubated for 24 hours under ambient conditions at 37 °c. after growing the bacteria, colony forming units (cfu) were counted and replanted to a new solid growth medium for further identification. identification of genera and species of bacteria was performed in accordance with the microbiological professional standards. moreover, chromogenic uriselect agar (biorad, redmond, usa), gram staining and additional enzymatic and biochemical tests have been performed, such as coagulase, catalase, bacitracin test, optochin test, novobiocin test for identifications of grampositive bacteria and oxidase, sugar fermentation with acid and gas production (triple sugar iron), test for indole production, h2s production, citrate utilization and urease test for identification of gram-negative bacteria. statistical analysis statistical analysis was performed using the spss software (ibm spss statistics ver. 16, ibm corporation, chicago, il) and microsoft excel. proportions were compared by chi-square test and fisher’s exact test. p values <0.05 were considered to be statistically significant. results in total, 739 bacterial cfu were grown with the mean value of 6.16 colonies in six species of bacteria: staphylococcus epidermidis, staphylococcus saprophyticus, streptococcus viridans, bacillus sp., klebsiella sp., neisseria sp. altogether, 36 samples (30%), 14 coins and 22 banknotes, were without the growth of bacteria, as shown in figure 1. on the coins of 1 and 2 hrk, bacteria were detected in 90% of cases (9/10), as opposed to 5 seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 7 southeastern european medical journal, vol 1, 2017. hrk coins, where bacteria were detected in 60% of cases (6/10). with regard to banknotes, on 500 hrk notes bacteria were detected in 80% of cases (8/10), as opposed to 20 hrk notes, where bacteria were detected in 40% of cases (4/10) (figure 2). all the banknotes and coins were taken from everyday life: there were nine banknotes which appeared almost “brand new” with the assumption that a minimum number of people touched them, as well as two coins and three banknotes that appeared extremely dirty. in total, there were 571 colonies on 38 coins, and 168 colonies on 46 banknotes isolated, which shows that some coins or banknotes had multiple colonies isolated. on the 10 lipa, 2 hrk and 5 hrk coins, s. epidermidis and bacillus sp. were isolated. along with the above listed bacteria, on the coin of 50 lipa, neisseria sp. was also isolated. s. epidermidis and s. viridans were found on the 20 lipa coin. it should be noted that on a single 20 lipa coin there were 201 colonies isolated, which was the maximum number of colonies found on a single coin. money with the most bacteria grown was 1 hrk, s. epidermidis, s. viridans, bacillus sp., neisseria sp. among banknotes, the most common bacterial species was s. epidermidis, found on notes of 10, 20, 50, 100, 200 and 500 hrk. bacillus sp. was also found on all of the listed notes, except 10 and 20 hrk. on the banknotes of 10 hrk, beside s. epidermidis, klebsiella sp., s. saprophyticus were also isolated. there is no statistical significance between the prevalence of bacterial contamination of banknotes and coins. the identified bacterial species are mostly part of the normal human flora. clinically relevant pathogenic, and potentially pathogenic bacterial species were not found on croatian banknotes and coins except forone colony of klebsiella sp. discussion six species of bacteria were grown in 739 bacterial cfu. almost 30% of the money was bacteriologically clean. there were no statistical differences between the prevalence of bacterial contamination of banknotes and coins. the most common bacteria isolated was s. epidermidis (86.33%) with statistical significance both on banknotes and coins. despite the expectation that money is highly contaminated with bacteria and fungi (3), in this experiment we have shown that 30% of croatian money was bacteriologically clean, which is in accordance with observations from an experimental bacterial survival study conducted in the netherlands (4). it was also expected that the banknotes or coins with lower denomination will have more contaminants compared to those with higher denomination, but that was not proven in our study. there is no statistical significance between the prevalence of bacterial contamination of banknotes and coins. identified bacterial species are mostly part of normal human flora. some studies have shown that copper seems to be a limiting factor for bacterial survival on coins (1, 5, 6, 7), but in our study no statistical significance was found between banknotes and coins. also, it was expected that lower denomination banknotes are used more frequently in daily life and would be more contaminated, as shown in a bangladesh study (8), but in our study no statistical significance was found between figure 1. the frequency of banknotes and coins with cultured bacteria (60 banknotes and 60 coins). 46 38 14 22 0 5 10 15 20 25 30 35 40 45 50 banknotes coins growth no growth seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 8 southeastern european medical journal, vol 1, 2017. higher and lower denominations of banknotes. in recent, well designed experimental research, whose objective was to ascertain the survival rates of some of the most important bacterial species, including nosocomial pathogens such as methicillin-resistant staphylococcus aureus (mrsa), extended spectrum beta-lactamase (esbl) producing escherichia coli, and vancomycin-resistant enterococci (vre), it has been shown that croatian kuna was the cleanest currency among those examined (4). furthermore, the croatian kuna was found to inhibit the growth of all of the multi-drug resistant bacteria tested, which was completely unexpected (4). other currencies had a tendency to grow colonies of different pathogens: cultures of the romanian leu yielded mrsa, vre and esbl producing e. coli, the canadian and us dollar only yielded mrsa; the euro only esblproducing e. coli, the indian rupee only vre, and table 1. number of colony forming units (cfu) on banknotes and coins. there were 120 pieces of banknotes and coins in total. bacterial species total cfu coins with growth banknotes with growth total positive (percentage) s. epidermidis 638 41 38 79 (66%) bacillus sp. 33 9 7 16 (13%) s. saprophyticus 6 0 1 1 (1%) s. viridans 41 2 0 2 (2%) neisseria sp. 20 2 0 2 (2%) klebsiella sp. 1 0 1 1 (1%) total 739 54 47 101 (84%) figure 2. distribution of bacteriologically positive and negative findings per denominations in hrk (10 per each banknote/coin, total 120) 8 2 7 3 5 5 6 44 6 8 2 6 4 9 1 9 1 8 2 7 3 7 3 0 1 2 3 4 5 6 7 8 9 10 growth no growth 500 kuna 200 kuna 100 kuna 50 kuna 20 kuna 10 kuna 5 kuna 2 kune 1 kuna 50lipa 20lipa 10lipa seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 9 southeastern european medical journal, vol 1, 2017. the croatian kuna did not yield any of the 3 microorganisms (4). this can be the result of the material of which banknotes are made, as polymer-based banknotes presented lower bacterial counts than cotton-based bank-notes (1,3). other studies have shown that 100% of currency notes from india (9, 10, 11), bangladesh (8,9), iraq (9,11), and ghana (9,11,12) were found to carry pathogenic or potentially pathogenic bacteria. in 1972, a study was conducted by abrams and waterman and it was found that 42% of paper money and 13% of coins collected from laboratory workers was contaminated by potential pathogens, such as s. aureus, e. coli, klebsiella sp., p. aeruginosa, and proteus mirabilis (6). studies which showed significant contamination of banknotes and coins of different currencies with pathogenic and potentially pathogenic bacteria have been conducted previously (3, 13). it was also shown that money can be a vector for the spreading of the most significant clinical bacterial isolates and also have an impact on the emergence of hospital infections (1). conclusion the identified bacterial species are mostly part of the normal human flora. pathogenic, and potentially pathogenic bacterial species were not found on croatian banknotes and coins, except for one colony of klebsiella sp. despite the resulting information from our research, hand hygiene should be implemented after each contact with money. acknowledgement results have been presented as a poster during a student scientific meeting held in sarajevo in february 2016 (samed 2016). disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. angelakis e, azhar ei, bibi f, alghamdi ak, ashshi am, elshemi ag. paper money and coins as potential vectors of transmissible disease. future microbiol 2014;9:249–61. 2. mayo han a, new oo h, phyu win p, aye t. contamination of currency notes with enteric bacterial pathogens. journal of diarrheal diseases research. 1989;7:92–9. 3. kuria jk, wahome rg, jobalamin m, kariuki sm. profile of bacteria and fungi on money coins. east afr med j 2009; 86:151-5. 4. gedik h, voss ta, voss a. money and transmission of bacteria. antimicrob resist infect control 2013;2:1. 5. tagoe d, baidoo s, dadzie i, ahator d. a study of bacterial contamination of ghanaian currency notes in circulation. the internet journal of microbiology [internet] 2009;8(2) [5 pages]. available from: http://ispub.com/ijmb/8/2/6218. [accessed 19th march 2017]. 6. abrams bi, waterman ng. dirty money. jama 1972; 219:1202-3. 7. espirito santo c, morais pv, grass g. isolation and characterization of bacteria resistant to metallic copper surfaces. appl environ microbiol 2010; 71:1341–48. 8. vriesekoop f, russell c, alvarez-mayorga b, aidoo k, yuan q, scannell a, et al. dirty money: an investigation into the hygiene status of some of the world’s currencies as obtained. foodborne pathog dis 2010;7:1497502. 9. augusto t, pessôa r, sanabani ss. molecular analysis of bacterial microbiota on brazilian currency note surfaces. int j environ res public health 2015;13276–88. 10. pradeep nv, anupama, marulasiddaiah bs, chetana m, gayathri p, maduri sn. microbial contamination of indian currency notes in circulation. journal of research in biology 2012; 2:377-82. 11. alemu a. microbial contamination of currency notes and coins in circulation: a potential public health hazard. biomedicine and biotechnology 2014; 2:46–53. 12. ahmed su, parveen s, nasreen t, feroza b. evaluation of the microbial contamination of seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 10 southeastern european medical journal, vol 1, 2017. bangladesh paper currency notes (taka) in circulation. advan biol res 2010; 4:266–71. 13. tolba o, loughrey a, goldsmith ce, mb h, millar bc. survival of epidemic strains of methicillin-resistant staphylococcus aureus on coins. am j infect control 2007; 35:342–6. seemedj 2018, vol 2, no. 2 theory of unpleasant symptoms 17 southeastern european medical journal, 2018; 2(2) review theory of unpleasant symptoms and concept of nursing support 1 štefica mikšić 1, marina bošnjaković 2, ivana simić 3, barica stanić 3, marina stanković 3, željka stojkov3 1 faculty of medicine, josip juraj strossmayer university of osijek, osijek, croatia 2 secondary medical school slavonski brod, slavonski brod, croatia 3 “dr josip benčević” general hospital, slavonski brod, croatia corresponding author: štefica mikšić, steficamiksic@gmail.com received: aug 16, 2017; accepted: mar 10, 2019; published: mar 31, 2019 keywords: theory, unpleasant symptoms, pain, dyspnea, healthcare abstract theory of unpleasant symptoms – tous – has been developing since the 1990s. tous is a theory in healthcare developed by healthcare workers and intended to be applied by them. according to this theory, multiple symptoms may occur simultaneously, interact with each other and be multiplicative. several symptoms have been presented, including also psychological, physiological and cognitive aspects of a patient’s personality. symptom models described in the tous are the nociceptive model of dyspnea, symptom interpretation model, model of chronic dyspnea and symptom management model. tous is the first model to describe multiple, mutually related symptoms. by studying this theory, it can be concluded that controlling a single symptom is beneficial for controlling other symptoms. tous views a set of occurring symptoms without isolating any of the symptoms, but allowing for each symptom to be monitored separately. the theory helps in planning nursing interventions and facilitates healthcare delivery in general. the purpose of this theory is to help healthcare workers understand the symptoms and the methods used for their management. tous has been developed through clinical research; it establishes a logical connection between the symptoms and extends theoretical knowledge by taking into account previous factors, experiences of single or multiple symptoms and their impact on performance. (mikšić š, bošnjaković m, simić i, stanić b, stanković m, stojkov ž. theory of unpleasant symptoms and concept of nursing support. seemedj 2018; 2(2); 17-22) seemedj 2018, vol 2, no. 2 theory of unpleasant symptoms 18 southeastern european medical journal, 2018; 2(2) introduction nursing is a profession and an established scientific discipline that helps people achieve a healthy and productive life, or, rather, enables them to cope with particular deficiencies, poor health, illnesses and injuries the best way possible by using skillbased knowledge (1). the goal of nursing is not only to treat ill people, but to maintain good health, aid relaxation and provide relief and comfort. every healthcare theory provides nurses with guidelines for providing healthcare and helping the patients. theory of unpleasant symptoms (tous) was first mentioned in 1995 and revised in 1997. it advocates that particular symptoms frequently occur simultaneously and proposes monitoring of multiple symptoms. application of the theory, combined with clinical observations and relevant literature, results in better understanding the patients experiencing those symptoms during their illness. to make the application of this theory as simple as possible in day-to-day work, it is necessary to study its core from the very beginnings, through its development, and up to contemporary experiences. this paper presents models of appearance of primary symptoms, such as pain and dyspnea, in chronically ill and oncological patients. mental, physical and situational factors, as well as the sense of empathy, affect the development of such symptoms (2). the theory itself is aimed at investigating how to reduce the above-mentioned symptoms in order to achieve improvements in patients’ quality of life (3). development and description of the theory theory of unpleasant symptoms (tous) started developing in the 1990s, when audrey gift and linda pugh, its authors, wrote a book on clinical healthcare titled “dyspnea and fatigue”. their work was first published in 1995, and revised in 1997 (3). tous is a theory in healthcare developed by healthcare workers and intended to be applied by them. according to this theory, multiple symptoms may occur simultaneously, interact with each other and be multiplicative. group of symptoms affecting one another is called a symptom cluster (4). by controlling one symptom, we can control all the other symptoms. appearance of symptoms is conditioned by physiological, psychological and environmental factors. the work of a nurse is based on monitoring symptoms for the purpose of diagnosing a disease as early as possible, but also to help patients reduce their health issues. to corroborate their theory, the authors used various scales while conducting their research and studying patients. some of those were visual analogue scale – vas (5), visual analogue scale dyspnea – vasd (6), numeric rating scale – nrs (7), mcgill pain questionnaire (8), brief fatigue inventory – bfi (9), memorial symptom assessment scale – msas (10) and symptom distress scale – sds (11). symptom models most symptom theories in medical literature mainly focus on the experience of symptoms, but not on their control. tous describes four symptom models. nociceptive model of dyspnea is determined by environmental factors corresponding individually to the severity of illness. it provides answers to healthcare delivery from a psychological perspective. symptom interpretation model requires knowledge, understanding and identification of symptoms. there are three main criteria in this model – input, interpretation and outcome. input needs to have a strong influence in order to encourage thinking about something different. interpretation relates to making a differentiation on the basis of experiential reasoning. comparison between interpretations of symptoms is made by contrasting the most severe symptoms to typical symptom patterns, which is, at the same time, the only way they can be interpreted correctly. outcome is the third and the most important aspect of this model, as it reveals how patients actually feel and what they are willing to accept to be able to receive help. through interpretation, patients decide how they will control the symptoms they experience. seemedj 2018, vol 2, no. 2 theory of unpleasant symptoms 19 southeastern european medical journal, 2018; 2(2) model of chronic dyspnea is the first model for monitoring changes in behaviour. its course is easier to follow using a longitudinal curve. initially, it suddenly increases, then it returns to its initial state, and with time it rises again and reaches its peak. the model consists of physiological factors, dyspnea and, finally, its consequences. chronic dyspnea is increased when influenced by episodes of acute dyspnea. fatigue, depression, irritability, fear, reduced physical activity and social isolation appear as consequences. symptom management model is the most effective model for symptom control. patients’ experience of symptoms, symptom control and outcome of symptoms are interconnected. symptoms are not controlled only by patients, but also by their families and healthcare workers. quality of life, emotional and functional status and morbidity in use of healthcare services depend on the outcomes of symptom control. this model is important because it can assist doctors and nurses in helping patients control the symptoms they experience. theory analysis comprehensibility of the theory of unpleasant symptoms is observed through description of definitions and technical terms. theorists have described techniques for overcoming unpleasant symptoms through clinical practice. all constituents of the theory, as well as key concepts, are described in a clear and understandable manner. symptom models have been described starting from simple to complex ones. the relationship between clinical practice, research and theory is understandable. it is clear that all models argue for existence of multiple symptoms and suggest that controlling one symptom helps control the other ones. use of key concepts both in explaining the theory and presenting the details of symptom models is also consistent. high level of consistency can be seen in the relationship between all the constituents, as all of them mutually support each other. the theory is applicable to all the patients experiencing the appearance of unpleasant symptoms. it was developed based on clinical practice and revised with the help of relevant literature. interpretation of symptoms is monitored and symptoms can be observed separately or collectively. numerous instruments are used for assessment of symptoms, such as the numeric rating scale (nrs), mcgill pain questionnaire (mpq), and brief fatigue inventory (bfi). there are also various scales for assessment of multiple symptoms, for instance the memorial symptom assessment scale and symptom distress scale. tous was developed with help from healthcare workers and their observations. according to its criteria, it is consistent with the middle-range theory. it is used to examine the relations between patients’ symptoms and their everyday cognitive functions. the theory can be used as an educational and a research tool in all areas and fields of healthcare. concept of nursing support the term support has multiple meanings and may signify some kind of help, support and relief. it is commonly used in the healthcare system without a clear description of what is meant by it. research of the term “support” in the context of nursing has resulted in a wide range of its definitions. authors ellis, jackson and stevenson state that dictionaries and related literature offer a wide range of meanings of the word, from “preventing a person to give up” to “encouraging statements” (12). by analysing those different meanings, we come to other related concepts such as help, safety, and empathy, which can be independently subjected to analysis of the concept, but in practice they are identical to the concept of support. support/care that nurses provide to a patient is undoubtedly an important factor of healthcare, but providing support is in fact very complex, because patients and nurses may, considering different predisposing factors, perceive support differently. some of those factors can be patients’ age and/or education or particular organisational factors, such as the ratio between nurses and hospitalised patients (13). providing support carries a different meaning for different people in different environments, but does not diminish its value. a seemedj 2018, vol 2, no. 2 theory of unpleasant symptoms 20 southeastern european medical journal, 2018; 2(2) nurse may think she is providing support, while the patient may at the same time, due to his/her upbringing, assume that it is the nurse’s duty to act in such manner only because she is required to do so. both male and female nurses should not have a fixed attitude in their relationship with patients and assume that what they provide is support. they should rather adopt a flexible approach and try to clarify, define and eventually provide a particular kind of support. built on an open and collaborative relationship, this kind of approach would ensure there are no ambiguities in its perception. measuring support is extremely complex because it is limited to a feeling expressed by a patient. any stay in hospital, regardless of the severity of symptoms, causes emotional difficulties, as well as anxiety and depression. moreover, each patient must go through all the stages of adaptation to a disease (shock, denial, bargaining, depression, adaptation) (14). in their day-to-day work, nurses notice the aforementioned difficulties – phenomena and, aside from physical help, they often provide patients with psychological, spiritual and social support. physical help can pertain to assistance with walking, while emotional support can refer to support provided when somebody is grieving. as already stated, the phenomena can be different, ranging from worry, fear and helplessness to physical limitations and cognitive impairments. when these phenomena are present, the patient also needs individual help from a nurse. nurses meet patients’ needs when they provide care using a holistic approach. in order for a nurse to cater to those needs in a satisfactory manner, it is necessary that both the nurse and the patient cooperate and negotiate on the type of help required by the patient. as a result, an important factor emerges in the process of providing healthcare, namely the need for specificity in determining a proper healthcare plan. as the actual purpose of nursing in this context is to create a common meaning for the term support, this is an extremely important factor for ensuring that healthcare is provided in a proper and satisfactory manner. connection between the theory of unpleasant symptoms and concepts of nursing support in practice male and female nurses are faced with increasingly complex requests to recognise and solve the patients’ issues. that requires solid theoretical knowledge applicable in practice, a holistic approach, experience, skills, competence, and motivation. application of practical clinical skills based on theory and relevant concepts is precisely what is unfailing in effectiveness. understanding their relations based on scientific facts and research conducted by nurses will create the preconditions for emergence of theories and concepts in nursing practice applicable to dayto-day nursing tasks. the opinion held by the author of the book nursing theories, j. b. george, is that concepts such as support produce nursing theories and that therefore, for some theories, a detailed description of a concept serves as the basis for its development (15). theory of unpleasant symptoms and concept of nursing support have a very high level of applicability. besides helping male and female nurses to use a comprehensive approach to symptom appearance and to plan nursing interventions, they also facilitate healthcare delivery in general. authors tested their theory on patients suffering from a malignant disease, patients with chronic pulmonary disease and patients in the terminal phase of lung cancer. in accordance with our previous work, we saw that the theory and the concept can also be applied to cardiac patients. in this case, it was applied to a patient suffering from an acute myocardial infarction. case report: patient n.n., aged 43, was admitted to the coronary unit of “dr. josip benčević” general hospital slavonski brod, diagnosed with an acute myocardial infarction. upon admission he was pale, his skin was cold, he was sweating, he was scared and expressed concern for his condition. he was complaining about the pain in his chest that was spreading to his shoulders and back and was suffering from shortness of breath. based on the above-mentioned, the following issues can be identified: seemedj 2018, vol 2, no. 2 theory of unpleasant symptoms 21 southeastern european medical journal, 2018; 2(2) ● pain – reaction to one’s physiological state. presence of other unpleasant symptoms also depends on the intensity of pain. ● shortness of breath (dyspnea) – increases as the pain increases. ● fear/concern affective reaction to current condition, “i am afraid i am going to die” nursing interventions were aimed at assessing the intensity and character of pain. on a scale from 0 to 10, the patient rated pain as 8 (vas pain scale). monitoring of vital functions, nasal catheter oxygenation and application of prescribed therapy were performed. during the nursing interventions, all the procedures were explained to the patient and nurses attempted to comfort him, i.e. to provide support. through the application of the prescribed therapy and through decreasing the intensity of the pain, dyspnea also decreased and the patient was less scared. from all of the above, it can be concluded that the theory of unpleasant symptoms and the concept of nursing support were both applied in this case. by affecting a physical symptom (pain decrease) and providing psychological support, other unpleasant symptoms that were present also decreased. discussion nursing theories represent a tool for development of knowledge and are not designed as a recipe applicable in practice. they should be developed in accordance with nurses’ creative thinking and their interaction with a patient (16). tous was created by taking the results obtained from practice and linking them to scientific literature. many authors in the nursing science have developed models of patient care based on the existing nursing theories. by analysing the theory according to the criteria established by mckenna, it was noted that this theory is applicable both in practice and in further research. the advantage of the tous is that it helps nurses identify the objectives of nursing practice, which are, in this case, to decrease unpleasant symptoms and monitor the effect of one symptom on the other, and thus improve the quality of healthcare. regarding the provision of nursing support of any kind, it is equally ambiguous as the term “providing care for” or “taking care of” a patient. support measurement and assessment in a healthcare plan is a very complex issue. in healthcare, the term “support” is often used without a clear description of what is actually meant by it. what we conceptualise as nursing support is the product of a relationship between a nurse and an individual within a particular context. different meanings arising from interpersonal communication exist simultaneously at different contextual levels. when it comes to providing support, the goal of nursing is to create a common meaning for the term “support”, which requires mutual cooperation and a flexible approach. by accepting the ambiguity in the perception of the term “support” or “assistance”, a nurse is able to cooperate with individuals and patients to create a common meaning for the word “support” in practice. in other words, together they can reach an understanding regarding what “support” actually means to that person. the concept of support as a method demonstrates the complexity of communication and different meanings that people may assign to it depending on the context and previous experiences. conclusion the theory of unpleasant symptoms (tous) is a middle-range theory, i.e. a healthcare theory intended to be applied by healthcare workers. it includes specific concepts and connects them. based on its interpretation, it can be concluded that unpleasant symptoms are interconnected and affected by various psychological, physical and situational factors. the work of a nurse is based on monitoring symptoms for the purpose of diagnosing a disease as early as possible, but also on helping patients reduce their health issues. this theory is acceptable because it helps in planning of nursing interventions and facilitates healthcare delivery in general. knowledge and skills gained through education and research are important to successfully perform nursing practice, achieve compliance with nursing principles and fulfil any seemedj 2018, vol 2, no. 2 theory of unpleasant symptoms 22 southeastern european medical journal, 2018; 2(2) expectations the community may have of male and female nurses, as well as the expectations they have of themselves based on their own professional ethics (17). an important aspect of the work of each nurse, as well as of healthcare in general, should be to provide patients with support, regardless of how complex that may be. tous and the concept of nursing support have a very high level of applicability. based on personal experience at our workplace, we have come to know that this theory and concept are also applicable to patients suffering from diseases other than those investigated in this paper. therefore, there is potential for their further development and research in nursing. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare references 1. 1. gavranić d., iveta v., sindi j. konceptualni modeli sestrinstva i načela sestrinske prakse. pregledni članak. 2016; 21: 148-52. 2. peterson s.j., bredow s.t. middle range theories. application to nursing research. 2nd ed. wolters kluwer/lippincott williams and wilkins. philadelphia; 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85(5):11861196. 10. portenoy r.k., thaler h.t., lepore j. m., friedlander-klar h., kiyasu e. the memorial symptoms assessment scale: an instrument for the evaluation of symptom prevalence, characteristics and distress. european journal of cancer, 1994; 30a(9): 1326-1336. 11. mccorkle r., young k. development of a symptom distress scale. cancer nursing, 1978; 1: 373-378. 12. allen r.e. oxford paper back dictionary. oxford university press, oxford 1990. 13. matilla e., kaunonen m., aalto p., ollikainen j., astedt-kurki p. support for hospital patients and associated factors. scand j caring sci. 2010; 24: 734-745. 14. havelka m. zdravstvena psihologija. zagreb: školska knjiga; 2009. 15. george j.b. nursing theories: the base for professional nursing practice. 6. izd. london: appleton& lange; 1995. 16. ljubičić m., šare s. povezanost teorije i prakse u zdravstvenoj njezi. pregledni članak. 2015; 20: 254-6. 17. mojsović z. sestrinstvo u zajednici. visoka zdravstvena škola. zagreb: 2005 seemedj 2019, vol 3, no. 1 significance of hba1c in monitoring diabetes 60 southeastern european medical journal, 2019; 3(1) original article significance of hba1c in monitoring diabetes at the public hospital, guyana 1 rajini kurup 1, michelle deoraj 1, sasha persaud 1 1 faculty of health sciences, university of guyana, guyana corresponding author: rajini kurup, rajini.kurup@uog.edu.gy received: october 6, 2018; revised version accepted: may 7, 2019; published: may 31, 2019 keywords: hba1c, guyana, diabetes abstract aim: to determine the prevalence of baseline/mean hba1c among patients with suspected diabetes using bio-rad for the first time at a public hospital in guyana. methods: a retrospective, laboratory-based, descriptive study examined 1,547 diabetic patients who underwent repeat hba1c testing at a public hospital laboratory in guyana between 2010 and 2014. all statistical analyses were performed using spss 21.0, jmp and microsoft excel. distributions were used to show frequencies, the bivariate fit test was done to achieve correlations and significance, the χ2 test was used to compare the differences in proportions and anova was used for differences in mean differences. results: a total of 1,547 patients were identified for the study. mean age and standard deviation (sd) was 59.9 ± 11.47 (95% ci 59.3 60.4). mean value for test 1 hba1c was 9.2 ± 2.7 (95% ci 9.1-9.3), test 2 hba1c recorded 9.1 ± 2.5 (95% ci 8.9-9.2), test 3 hba1c was 9.6 ± 2.6 (95% ci 9.3-9.9) and test 4 hba1c recorded a mean of 9.6 ± 2.6 (95% ci 9.0-10.2). conclusions: the mean hba1c of the diabetic population was observed to be higher than the baseline in all tests. (kurup r, deoraj m, persaud s. significance of hba1c in monitoring diabetes at the public hospital, guyana. seemedj 2019; 3(1); 60-68) seemedj 2019, vol 3, no. 1 significance of hba1c in monitoring diabetes 61 southeastern european medical journal, 2019; 3(1) introduction diabetes mellitus is defined by the world health organization (who) as a chronic disease caused by inherited and/or acquired deficiency in the production of insulin by the pancreas (1). it is estimated that 39 million people are living with diabetes in the north america and the caribbean (nac) region (2). in 2014, there were 61,800 cases of diabetes in guyana and diabetes was the cause of 1,025 deaths in adults aged 20-79. the international diabetes federation (idf) also estimated that there are about 15,400 undiagnosed cases of diabetes (3). with the prevalence of diabetes increasing at an alarming rate both nationally and internationally, diabetes-related complications and deaths tend to increase as well (4-8). it is therefore critical to maintain good glycemic control to keep diabetes in check and reduce diabetes-related complications (9). hba1c has been recommended as a test option for the diagnosis of diabetes by both the american diabetes association (10) and the world health organization (who) (11). both organizations advised that after a result consistent with the diagnosis of diabetes (≥ 6.5% (48 mmol/mol)), testing of hba1c levels should be repeated in asymptomatic patients within 2 weeks to rule out the rare occurrence of a sample being mislabeled. not much research has been conducted in the area of repeat testing of hba1c; however, driskell et al. (2014) stated that “the optimal testing frequency required to maximize the downward trajectory in hba1c was four times per year, particularly in those with an initial hba1c of ≥7% (≥53 mmol/mol), supporting international guidance. testing 3-monthly was associated with a 3.8% reduction in hba1c compared with a 1.5% increase observed with annual testing; testing more frequently provided no additional benefit. compared with annual monitoring, 3-monthly testing was associated with a halving of the proportion showing a significant rise in hba1c (7–10 vs. 15–20%)” (12). the authors concluded that monitoring frequency is associated with a significant detrimental effect on diabetes control and that to achieve the optimum downward trajectory in hba1c, monitoring frequency should be quarterly. there are also reports that suggest that guidelines for testing hba1c are not necessarily being followed (13-15). this study therefore evaluated for the first time the baseline/mean hba1c (mmol/mol) among the guyanese population and aims to determine if a standard international algorithm has been followed in regard to repeat testing of patients with suspected diabetes and of undiagnosed/highrisk population. methods this was a laboratory-based retrospective study conducted between 2010 and 2014 at a level iii public hospital in guyana. all patients who had already performed the hba1c test to screen for diabetes and those who had complete information were included in the study. any patient who did not follow the hba1c algorithm was excluded. inappropriate ordering of tests was defined as any order for testing a given patient occurring more than 3 months after the previous order. bio-rad d-10 was used to monitor hba1c at the public hospital. bio-rad d10 utilizes the principle of high performance liquid chromatography (hplc) to determine the percentage of hba1c. permission to conduct this research was obtained from the ethics committee under the ministry of health, guyana, and from the director of medical services of the public hospital. data analysis the data collected from the database were first entered into a spreadsheet using microsoft excel. all statistical analyses were performed using spss 21.0, jmp and microsoft excel. distributions were used to show frequencies, the bivariate fit test was done in order to achieve correlations and significance, the χ2 test was used to compare the differences in proportions and anova was used for differences in mean differences. a p-value of < 0.05 was used to show significance. seemedj 2019, vol 3, no. 1 significance of hba1c in monitoring diabetes 62 southeastern european medical journal, 2019; 3(1) results a total of 1,547 eligible patients were selected for the study from the database of the gphc medical laboratory. the patient population analysis had the following baseline characteristics: mean age was 59.9 ± 11.5 years (95% ci 59.3 60.4), 5% of the patients were between the ages of 21 and 40, 41% were between the ages of 41 and 60 and 54% were between the ages of 61 and 90. figure 1 shows the hba1c percentage in test 1, the maximum, median and minimum values of 19.2%, 8.9% and 4.2% respectively and a mean and standard deviation of 9.2 ± 2.7. for the hba1c percentage in test 2, the maximum, median and minimum values were 18.5%, 8.7% and 4.0% respectively, with a mean (sd) of 9.1 ± 2.5. for test 3, 396 patients were tested with the maximum, median and minimum values of 19.7%, 9.4% and 4.1% respectively, and a mean (sd) of 9.6 ± 2.6. only 82 patients were tested for a fourth time, with the maximum, median and minimum values of 15.1%, 9.2% and 5.2% respectively, and a mean (sd) of 9.6 ± 2.6 (figure 1). figure 1. distribution of hba1c test results the mean hba1c ± standard deviation (sd) of all tests is shown in table 1. test 1 had a mean of 9.23 ± 2.6 (95% ci 9.1-9.4), test 2 had a mean of 9.08 ± 2.5 (95% ci 8.95-9.20), test 3 had a mean of 9.59 ± 2.6 (95% ci 9.34-9.85) and test 4 had a mean of 9.59 ± 2.6 (95% ci 9.01-10.17). test 3 hba1c % test 4 hba1c % test 1 hba1c % test 2 hba1c % seemedj 2019, vol 3, no. 1 significance of hba1c in monitoring diabetes 63 southeastern european medical journal, 2019; 3(1) table 1. mean ± sd of hba1c in regard to gender hba1c male 95% ci female 95% ci total 95% ci mean ± sd mean ± sd mean ± sd test 1 8.9 ± 2.7 8.7-9.2 9.4 ± 2.6 9.5-9.2 9.2 ± 2.7 9.1-9.4 test 2 8.7 ± 2.4 8.5-8.9 9.2 ± 2.6 9.4-9.1 9.1 ± 2.5 8.9-9.2 test 3 9.2 ± 2.5 8.7-9.7 9.8 ± 2.6 9.5-10.1 9.6 ± 2.5 9.3-9.8 test 4 8.8 ± 2.5 7.8-9.8 9.9 ± 2.7 9.3-10.7 9.5 ± 2.6 9.0-10.2 the correlation analysis between each test is shown in figure 2. the correlation between test 1 and test 2, test 3 and test 4 was 0.64 (95% ci 0.61-0.67), 0.58 (95% ci 0.50-0.64) and 0.66 (95% ci 0.52-0.77), respectively. the correlation between test 2 and test 3 was 0.64 (95% ci 0.570.69), and the one between test 2 and test 4 was 0.66 (95% ci 0.51-0.76). the correlation between test 3 and test 4 was 0.64 (95% ci 0.490.75). figure 2. correlation between individual hba1c test values seemedj 2019, vol 3, no. 1 significance of hba1c in monitoring diabetes 64 southeastern european medical journal, 2019; 3(1) mean ± sd of each hba1c test among male and female patients is shown in table 2. mean hba1c was higher among females in all four hba1c tests. females were more at risk and all four tests indicated that females were above the and p < 0.05)). table 2. prevalence of mean hba1c and risk factor among male and female population female male total hba1c > mean n (%) n (%) p-value rr (95% ci) n (%) 95% ci test 1 no 145 (9.4) 92 (0.1) < 0.05 1.13 (1.02-1.26) 237 (15.3) 13.6-17.2 yes 906 (58.6) 404 (26.1) 1310 (84.7) 82.8-86.4 test 2 no 147 (9.5) 85 (0.1) < 0.05 1.04 (0.99-1.08) 232 (15) 13.3-16.9 yes 904 (58.4) 411 (26.6) 1315 (85.0) 83.1-86.7 test 3 no 27 (6.8) 15 (3.8) > 0.05 1.04 (0.96-1.13) 42 (10.5) 7.9-13.9 yes 258 (64.7) 99 (24.8) 357 (89.5) 86.1-92.1 test 4 no 3 (3.7) 2 (2.4) > 0.05 1.03 (0.90-1.16) 5 (6.1) 2.6-13.5 yes 53 (64.6) 24 (29.3) 77 (93.9) 86.5-97.4 the mean difference ± standard error (se) change between two hba1c tests is shown in table 3. the duration between individual tests is shown in figure 3. mean duration between test 1 and test 2 was 16.14 ± 11.3 (95% ci 15.6-16.7), with the standard error of 0.28; for test 2 and test 3, it was 12.43 ± 8.83 (95% ci 11.6-13.3), with an se of 0.44; for test 3 and test 4, it was 10.25 ± 8.8 (95% ci 8.32-12.2), with an se of 0.96. figure 3. duration and standard error between individual tests seemedj 2019, vol 3, no. 1 significance of hba1c in monitoring diabetes 65 southeastern european medical journal, 2019; 3(1) table 3. correlation and mean difference between each test hba1c mean difference se 95% ci r test 1 test 2 -0.15 0.05 -0.26-0.04 0.64 test 1 test 3 0.06 0.12 -0.17-0.29 0.57 test 1 test 4 0.22 0.23 -0.25-0.68 0.67 test 2 test 3 0.11 0.10 -0.10-0.32 0.63 test 2 test 4 -0.23 0.24 -0.70-0.23 0.67 test 3 test 4 -0.35 0.24 -0.83-0.12 0.65 of the 1,547 patients included in the study, for 828 (53.5%) patients, the hba1c result decreased in test 2 when compared to test 1. of the 396 patients who were tested for a third time, the hba1c result decreased for 171 (43.2%) patients when compared to test 2. of the 83 patients tested for a fourth time, 46 (56%) patients also experienced a decrease. discussion the main outcome was the proportion of patients undergoing repeat hba1c testing and the proportion of patients whose baseline hba1c value decreased. this study used the hba1c threshold for the diagnosis of diabetes (48 mmol/mol (6.5%)), according to the new diagnostic criteria for diabetes adopted by the american diabetes association (16). this study represents the first attempt to review and analyze the hba1c data of patients using bio-rad at the georgetown public hospital corporation. older patients, between the ages of 61 and 90, represented the largest group (54%) in the diabetic population and female patients were above baseline hba1c in more cases than male patients. a similar pattern has been noted in other studies, where female patients of all age groups and races were found to be at risk (17). this study revealed that no standard guidelines were followed by the clinicians in terms of repeat testing intervals. there were many variations noted in regard to the duration between tests. discordant hba1c results have created confusion among the clinicians. clinicians need to be aware that significant testretest variation exists in hba1c and is sometimes quite large (18). mean hba1c for all four tests was recorded above 9.0, which is much greater than the baseline value. many studies have warned that hba1c concentrations above 48 mmol/mol (6.5%) are risk factors for development of diabetic retinopathy, macrovascular outcomes and death (18-21). the international expert committee (iec) (24) and ada (16) have proposed new diagnostic criteria for diabetes on the basis of hba1c measurement, in which hba1c of ≥ 6.5% is defined as diabetes. in the present study, an hba1c level of 6.5% had a reasonably high specificity (99.1%) and low false-positive rate (0.9%) for the diagnosis of diabetes, which is in complete concordance with iec and ada recommendations. repeat testing showed a significant decrease in hba1c levels in a portion of patients. a decrease in a follow-up test is favored because this is indicative of following proper treatment strategies and overall control of the patient’s diabetic condition. it is also understood that a decrease should not be expected in every case of repeat testing, since an increased value maybe be accounted for by a number of different reasons. multiple factors affect the accuracy of hba1c as an indicator of average seemedj 2019, vol 3, no. 1 significance of hba1c in monitoring diabetes 66 southeastern european medical journal, 2019; 3(1) glucose concentration, including abnormal erythrocyte lifespan, assay-related artifacts, fast vs. slow glycosylation, ethnicity, pregnancy, use of drugs and acute illness. different factors affect the validity of hba1c, such as iron deficiency, altered hemoglobin structure, erythrocyte lifespan and interracial variability, age (the elderly and children), gender and pregnancy (25, 26). it is a well-known fact that high levels of hba1c are associated with an increased risk of atrial tachyarrhythmia and paroxys¬mal atrial fibrillation in patients with type 2 dm (27). furthermore, it has been reported that an increase of 1% in hba1c concentration was associated with roughly a 30% increase in allcause mortality among diabetes patients (28). this shows that the proportion of hba1c percentage increase is far too high and, as such, stricter measures should be implemented to reduce this percentage. conclusion it is a fact that hba1c is a test that is accurate and easy to administer for diagnosing diabetes, especially in low and middle income countries like guyana. however, the mean hba1c percentage of the diabetic population in guyana was found to be 9.4%, which is far higher than ada’s established hba1c criteria for prediabetes and diabetes (5.7% and 6.5%). therefore, the researchers recommend that strict guidelines be followed by physicians in regular and accurate testing of hba1c. acknowledgments sincere thanks to dr. pheona mohamed and ms. marinda leich for providing all the necessary facilities for this research. references 1. world health organisation. 2015. diabetes mellitus. accessed 01 15, 2015. http://www.who.int/mediacentre/fa ctsheets/fs138/en/. 2. national diabetes information clearinghouse. 2015. diagnosis of diabetes and prediabetes. 09 10. accessed 02 05, 2015. http://diabetes.niddk.nih.gov/dm/p ubs/diagnosis/. 3. international diabetes federation. 2014. idf diabetes atlas sixth edition. accessed 02 03, 2015. http://www.idf.org/sites/default/fil es/da-regional-factsheets2014_final.pdf. 4. mokdad ah, ford es, bowman ba, dietz wh, vinicor f, bales vs, et al. prevalence of obesity, diabetes, and obesity-related health risk factors. jama 2003; 289: 76–9. 5. wild s, roglic g, green a, sicree r, king h. global prevalence of diabetes: estimates for the year 2000 and projections for 2030. diabetes care 2004; 27:1047–53. 6. danaei g, finucane mm, lu y, singh gm, cowan mj, paciorek cj, et al. national, regional, and global trends in systolic blood pressure since 1980: systematic analysis of health examination surveys and epidemiological studies with 786 country-years and 5•4 million participants. lancet 2011; 378: 31–40. 7. whiting dr, guariguata l, weil c, shaw j. idf diabetes atlas: global estimates of the prevalence of diabetes for 2011 and 2030. diabetes res clin pract; 2011: 94: 311–21. 8. kanavos p, van den aardweg s. diabetes expenditure, burden of disease and management in 5 eu countries. lse health, london school of economics. seemedj 2019, vol 3, no. 1 significance of hba1c in monitoring diabetes 67 southeastern european medical journal, 2019; 3(1) 9. nathan dm, buse jb, davidson mb, heine rj, holman rr, sherwin r, et al. management of hyperglycemia in type 2 diabetes mellitus: a consensus algorithm for the initiation and adjustment of therapy. update regarding the thiazolidinediones. diabetologia 2008; 51: 8–11. 10. american diabetes association. diagnosis and classification of diabetes mellitus. diabetes care 2010; 33: s62 s69. 11. world health organization. use of glycated haemoglobin (hba1c) in the diagnosis of diabetes mellitus. geneva, who press, 2011. 12. driskell oj, holland d, hanna fw, jones pw, pemberton rj, tran m, et al. inappropriate requesting of glycated hemoglobin (hba1c) is widespread: assessment of prevalence, impact of national guidance, and practice-to-practice variability. clin chem 2012; 58: 906– 915. 13. salvagno gl, lippi g, targher g, montagnana m, guidi gc. monitoring glycaemic control: is there evidence for appropriate use of routine measurement of glycated haemoglobin? clin chem lab med 2007;45: 1065–1067. 14. loh tp, tan km, saw s, sethi sk. glycated haemoglobin: what is the diagnostic yield at shortened testing intervals? diabetes res clin pract 2011: 94: e40–42. 15. lyon aw, higgins t, wesenberg jc, tran dv, cembrowski gs. variation in the frequency of hemoglobin a1c (hba1c) testing: population studies used to assess compliance with clinical practice guidelines and use of hba1c to screen for diabetes. j diabetes sci technol 2009; 3(3): 411– 417. 16. american diabetes association. diagnosis and classification of diabetes mellitus. diabetes care 2010; 33: s62–s69. 17. zhao w, katzmarzyk pt, horswell r, wang y, johnson j, hu g. sex differences in the risk of stroke and hba1c among diabetic patients. diabetologia 2014; 57(5):918-926. 18. mcdonald tj and warren r. diagnostic confusion? repeat hba1c for the diagnosis of diabetes. diabetes care 2014: 37: e135–e136 | doi: 10.2337/dc14-0055 19. colagiuri s, lee cmy, wong ty, balkau b, shaw je, borch-johnsen k, et al. glycemic thresholds for diabetes specific retinopathy: implications for diagnostic criteria for diabetes. diabetes care 2011; 34: 14550. 20. khaw kt, wareham n, bingham s, luben r, welch a, day n. association of hemoglobin a1c with cardiovascular disease and mortality in adults: the european prospective investigation into cancer in norfolk. ann intern med 2004; 141: 413-20. 21. selvin e, steffes mw, zhu h, matsushita k, wagenknecht l, steffes mw, et al. glycated hemoglobin, diabetes, and cardiovascular risk in nondiabetic adults. n engl j med 2010; 362: 800-11. 22. stratton im, adler ai, neil haw, mathews dr, maley se, cull ca, et al. association of glycaemia with macrovascular and microvascular complications of type 2 diabetes (ukpds 35): prospective observational study. bmj 2000; 321: 405-12. 23. emden m. glycated haemoglobin for the diagnosis of diabetes. aust prescr 2014; 37: 98–100. seemedj 2019, vol 3, no. 1 significance of hba1c in monitoring diabetes 68 southeastern european medical journal, 2019; 3(1) 24. international expert committee report on the role of the a1c assay in the diagnosis of diabetes. diabetes care 2009; 32: 1327– 1334. 25. samir m and mordes jp. "the implications of using hemoglobin a1c for diagnosing." american journal of medicine 2011; 395-401. 26. juarez dt, demaris km, goo r, mnatzaganian cl, wong smith h. "significance of hba1c and its measurement in the diagnosis of diabetes mellitus: us experience." diabetes metab syndr obes: targets and therapy 2014; 7: 487-494. 27. lu zh, liu n, bai r, et al. hba1c levels as predictors of ablation outcome in type 2 diabetes mellitus and paroxysmal atrial fibrillation. herz. 2015; 40:130–6. 28. khaw kt, wareham n, luben r, et al. glycated haemoglobin, diabetes, and mortality in men in norfolk cohort of european prospective investigation of cancer and nutrition (epicnorfolk). bmj. 2001; 322:15–8. seemedj 2018, vol 2, no. 1 impact of delivery room resuscitation efforts 29 southeastern european medical journal, 2018; 2(1) impact of delivery room resuscitation efforts on admission temperatures in infants born < 32 weeks gestation darjan kardum1, boris filipović-grčić2, andrijana muller3, damir lončarević4 1 neonatal intensive care unit, department of pediatrics, university hospital osijek, faculty of medicine osijek, josip juraj strossmayer university in osijek, osijek, croatia 2 neonatal intensive care unit, department of pediatrics, university hospital centre zagreb, zagreb, school of medicine, university of zagreb, zagreb, croatia 3 department of gynecology and obstetrics, university hospital osijek, faculty of medicine osijek, josip juraj strossmayer university in osijek, osijek, croatia 4 neonatal intensive care unit, department of gynecology and obstetrics, university hospital centre zagreb, zagreb, croatia corresponding author: darjan kardum – darjankardum@gmail.com received: october 30, 2017; revised version accepted: april 5, 2018; published: november 27. 2018 keywords: premature infants, admission hypothermia abstract aim: this study aimed to determine how delivery room resuscitation efforts influence admission temperatures in premature infants born before 32 weeks gestation. methods: we retrospectively analyzed a cohort of premature infants born before 32 weeks gestation from january 2014 until december 2016. we compared the impact of resuscitation efforts performed in the delivery room on the admission temperature. hypothermia was defined as a core temperature of less than 36.5°c on admission. the primary outcome was admission temperature in the neonatal intensive care unit. secondary outcomes were apgar scores in the first and fifth minute, ph on admission, respiratory distress syndrome requiring surfactant, persistent ductus arteriosus, necrotizing enterocolitis, late onset sepsis, kidney failure, intraventricular hemorrhage and death before hospital discharge. exclusion criteria were inevitably lethal congenital malformations. results: we studied 147 infants born < 32 weeks gestation. in the delivery room, 66 (44.8%) of infants were given standard thermal care, 20 (13.6%) received standard care and continuous positive airway pressure (cpap), 49 (33.3%) received standard care and ippv, whereas 12 (8.1%) of infants received standard care and extensive resuscitation efforts (intubation and/or chest compressions and/or epinephrine). patients receiving standard care and intermittent positive-pressure ventilation (ippv) had significantly lower admission temperatures than those given standard care only (35.7°c vs. 36.2 °c, p < 0.02). no correlation was found in infants receiving cpap or extensive resuscitation efforts compared to those receiving standard thermal care only. conclusion: in our study, admission hypothermia was associated with ippv in the delivery room. application of cpap or extensive resuscitation efforts in the delivery room did not influence admission temperatures. (kardum d, filipović-grčić b, muller a, lončarević d. impact of delivery room resuscitation efforts on admission temperatures in infants born < 32 weeks gestation. seemedj 2018; 2(1); 29-35) seemedj 2018, vol 2, no. 1 impact of delivery room resuscitation efforts 30 southeastern european medical journal, 2018; 2(1) introduction admission hypothermia is a common problem in preterm neonates and is reported in up to 78% of preterm neonates admitted to neonatal intensive care units (1). the world health organization classifies 36.0°c to 36.4°c as cold stress or mild hypothermia, 32.0°c to 35.9°c as moderate hypothermia, and lower than 32.0°c as severe hypothermia (2). admission hypothermia is associated with increased mortality in premature neonates (3), higher rates of late onset sepsis (4), respiratory distress syndrome (5) and intraventricular hemorrhage (6). for this reason, multiple strategies for preventing admission hypothermia have been proposed: the use of polyethylene wraps, heated mattresses, chemical warming packs and humidified and heated respiratory gases (7, 8). miller et al. (9) reported decreased odds of hypothermia following no resuscitative efforts with the conclusion that this is due to the fact that very low birth weight infants are healthier and better able to maintain normothermia. on the other hand, lyu et al. (3) reported that resuscitation in the first 30 minutes after birth was associated with increased admission temperature. they speculate "that an infant requiring resuscitation is likely to be sicker and require more care, which should result in the care team ensuring that all practices are followed, including attending to body temperature" (3). this study aimed to analyze the impact of delivery room resuscitation efforts on admission temperatures in infants born < 32 weeks of gestation as well as short-term outcomes of these infants. materials and methods this retrospective cohort study was performed at a single, referral level iii nicu of university hospital osijek (osijek, croatia). the medical charts of all infants of gestational age at birth ≥ 22 + 0/7 – 31 + 6/7 weeks born from january 2014 to december 2016 were retrospectively reviewed. infants with major congenital anomalies were excluded. the study was approved by university hospital osijek ethical board. standard thermal care was provided for all infants immediately after birth, and included placing the infant in polyethylene wrap under a radiant warmer. the infants were divided in four groups with regards to resuscitation efforts undertaken. infants who received only standard thermal care were assigned to "none" group, those who received standard care and continuous positive airway pressure (cpap) were assigned to "cpap" group, if intermittent positive pressure ventilation (ippv) was applied (bag mask or t – piece), infants were assigned to the "ippv" group and if at any time extensive resuscitation efforts (chest compression and/or epinephrine and/or delivery room intubation) were performed, infants were assigned to "cei" group. outcomes were compared among groups. our nicu was next to the delivery room and the transport time was within 5 minutes. the first temperature on admission was measured by the rectal method and was recorded immediately after admission to the nicu. we collected data regarding intrapartum and demographic variables. antenatal steroids were defined as any doses of corticosteroids given before delivery. chorioamnionitis was defined upon pathohistological analysis of the placenta. a low apgar score was defined as an apgar score < 7. the short-term outcome variables included: respiratory distress syndrome (rds) that required surfactant therapy, necrotizing enterocolitis at bell’s stage ≥ ii, severe intraventricular hemorrhage (grades iii or iv), patent ductus arteriosus requiring treatment, late onset sepsis, kidney failure and death before hospital discharge. respiratory distress syndrome (rds) was diagnosed by clinical and radiographic findings, and surfactant was delivered according to croatian recommendations (10). infants were diagnosed with sepsis if they had positive blood cultures for either bacteria or fungi. severe intracranial hemorrhage was classified as ivh grade iii – iv as classified by papile et al. (11). necrotizing seemedj 2018, vol 2, no. 1 impact of delivery room resuscitation efforts 31 southeastern european medical journal, 2018; 2(1) enterocolitis (nec) was diagnosed in the presence of at least intestinal pneumatosis and/or portal venous gas (bell’s stage ≥2) (12). kidney failure was diagnosed if the infant met the neonatal rifle criteria for kidney failure (13). neonatal mortality was defined as infant death before hospital discharge. statistical analysis data are presented as arithmetic means, quartiles and standard deviations for continuous variables. categorical variables were presented with frequency tables and cross tables. due to the fact that variables were not normally distributed and some samples were small, nonparametric tests were used. for testing differences in distributions for two variables mann-whitney test was used and, in the case of three or more variables, kruskal-wallis anova was used. for testing dependence between two s were used. for the purposes of our study, p value of < 0.02 was considered to be statistically significant. statistical analyses were performed by using statistical software statistica 13.3. results during the 3-year period, 147 infants met the inclusion criteria for the study. the maternal and infant variables of the four groups are listed in table 1. compared to "none" group, the infants in the "ippv" group had lower birth weight (925g vs. 1207g, p < 0.02), and were more often born via c section (89.8% vs. 66.1%, p <0.02). compared to the "none" group, the infants in the "cie" group had lower birth weight (898 g vs. 1207g, p < 0.02), and were more often born following chorioamnionitis (96.1% vs. 46.9%, p <0.02). table 1. comparison of maternal and infant variables among groups ano statistically significant p value: "cpap" group compared to "none" group bp value: "ippv" group compared to "none" group cp value: "cie" group compared to "none" group the median admission temperature was 36.3 °c (iqr range 35.9 °c – 36.7 °c). admission hypothermia was found in 61.2% of the study population. fifty-seven (38.7%) infants were normothermic, 51 (34.6%) were mildly hypothermic and 39 (26.5%) infants were moderately hypothermic. there were no severely hypothermic infants. the highest admission temperature was found in premature infants with completed 30 weeks of gestation (36.5 °c) (fig 1). compared to the "none" group, the "ippv" group had lower admission temperatures (35.7 °c vs. 36.2 °c, p < 0.02). "cpap" and "cie" groups were not associated with higher rates of admission hypothermia compared to "none" group (table 2). compared to "none" group, infants in the "cpap" group had fewer apgar scores < 7 in the first (0%, vs. 30.3%, p <0.02) and fifth minute (0% vs. 25.7%, p <0.02) and lower mortality before discharge (0% vs. 18.1%, p < 0.02). "none" (n=66) "cpap" (n=20)a "ippv" (n=49) p valueb "cie" (n=12) p valuec gestational age (weeks), median (iqr) 29.5 (27.1 30.5) 29.6 (28.7 30.9) 27.5 (25.7 30.0) ns 27.0 (24.9 28.5) ns birth weight, (g) median (iqr) 1207 (872 – 1545) 1323 (966 – 1422) 925 (697 – 1186) <0.02 898 (721 1138) <0.02 female 33 (50%) 7 (35%) 27 (55.1%) ns 2 (16.6%) ns chorioamnionitis 31 (46.9%) 6 (30%) 25 (51.1%) ns 11 (91.6%) <0.02 maternal hypertension 14 (21.2%) 6 (30%) 10 (20.4%) ns 0 (0%) ns c section 44 (66.1%) 17 (85.1%) 44 (89.8%) <0.02 6 (50%) ns antenatal steroids 42 (63.6%) 15 (75%) 35 (71.4%) ns 4 (33.3%) ns seemedj 2018, vol 2, no. 1 impact of delivery room resuscitation efforts 32 southeastern european medical journal, 2018; 2(1) fig 1. admission temperatures (degrees centigrade) and gestational age table 2. admission temperature and outcomes among groups ivh, intraventricular hemorrhage; nec, necrotizing enterocolitis; pda, patent ductus arteriosus; rds, respiratory distress syndrome; los, late onset sepsis ap value in the "cpap" group compared to "none" group bp value in the "ippv" group compared to "none" group cp value in the "cie" group compared to "none" group 32 33 34 35 36 37 38 22 23 24 25 26 27 28 29 30 31 °c completed weeks of gestation "none" (n=66) "cpap" (n=20) p valuea "ippv" (n=49) p valueb "cie" (n=12) p valuec admission temperature, mean ± sd (°c) 36.2±0.77 36.4± 0.49 ns 35.9±0.69 <0.02 35.7±1.44 ns 1-min. apgar score < 7 20 (30.3%) 0 (0%) <0.02 31 (61.2%) <0.02 11 (91.6%) <0.02 5-min. apgar score < 7 17 (25.7%) 0 (0%) <0.02 26 (53.0%) <0.02 9 (75%) <0.02 ph on admission, mean ± sd 7,24 ± 0.1 7.24 ± 0.9 ns 7.15 ± 0.13 <0.02 7.1 ± 0.16 <0.02 rds requiring surfactant 51 (77.2%) 11 (55.0%) ns 41 (83.6%) ns 11 (91.6%) ns pda 8 (12.1%) 1 (5%) ns 4 (8.1%) ns 4 (33.3%) ns nec ≥2 grade 13 (19.7%) 3 (15%) ns 10 (20.4%) ns 7 (58.3%) <0.02 los 28 (42.4%) 7 (35.0%) ns 25 (51.0%) ns 9 (75.0%) ns kidney failure 7 (10.6%) 0 (0%) ns 5 (10.2%) ns 4 (33.3%) ns ivh ≥ grade iii 16 (24.2%) 3 (15%) ns 15 (30.6%) ns 4 (33.3%) ns mortality before discharge 12 (18.1%) 0 (0%) <0.02 19 (38.7%) ns 7 (58.3%) <0.02 seemedj 2018, vol 2, no. 1 impact of delivery room resuscitation efforts 33 southeastern european medical journal, 2018; 2(1) compared to "none" group, infants in the "ippv" group had more apgar scores < 7 in the first (61, 2%, vs. 30.3%, p <0.02) and fifth minute (53.0% vs. 25.7%, p <0.02) and lower ph values (7.15 vs. 7.24) but did not have lower mortality and morbidity. compared to the "none" group, the "cie" group had more apgar scores < 7 in the first (91.6%, vs. 30.3%, p <0.02) and fifth minute (75.0% vs. 25.7%, p <0.02) and lower ph values (7.11 vs. 7.24). they also exhibited higher incidence of nec grade ≥ 2 (58.3% vs. 19.7%, p <0.02) and higher mortality before discharge (58.3% vs. 18.1%, p <0.02). discussion this study aimed to analyze the impact of delivery room resuscitation efforts on admission temperatures in infants born < 32 weeks of gestation as well as short-term outcomes in infants treated in a croatian neonatal intensive care unit, using the who classifications of hypothermia: mild hypothermia, 36.0 to 36.4 1°c; moderate hypothermia, 32.0 to 35.9 °c; and severe hypothermia, below 32 °c (2). hypothermia rates in our cohort are similar to those reported in other studies (3, 14, 15). in our study, lower admission temperatures were significantly associated with intermittent positive pressure ventilation (ippv) but were not associated with application of continuous positive airway pressure (cpap) or extensive resuscitation efforts which include delivery room intubation, epinephrine administration or chest compressions. the finding that cpap application in the delivery room does not lead to increased hypothermia is consistent with miller et al., who speculate that very low birth weight are healthier and better able to maintain normothermia (9). this is also reflected in the finding that these infants had significantly better apgar scores in the first and fifth minute and had a low mortality before discharge. also, this is consistent with previous findings that delivery room application of cpap is beneficial regarding outcomes in premature infants (16, 17). on the other hand, the findings that extensive resuscitation efforts which include delivery room intubation, epinephrine administration or chest compressions do not lead (to a statistically significant extent) to lower rates of admission hypothermia are consistent with findings of lyu et al. (3), who demonstrated that admission hypothermia is not associated with extensive resuscitation efforts despite the fact that these infants are sicker and in poor condition. this is consistent with our findings that infants who require extensive resuscitation efforts have significantly lower birth weights, lower apgar scores and lower ph values on admission and have a high rate of death before discharge. compared to infants who received standard thermal delivery room care, infants who received ippv in the delivery room had lower admission temperatures. we speculate that there are several factors leading to this. these infants had significantly lower birth weight than the group that received only standard thermal care, but, on the other hand, this did not influence admission temperature in the extensive resuscitation group. we speculate that lower admission temperature in the ippv group could be the result of ventilating these infants with unheated gases. duration of resuscitation must also be taken into consideration. this is one of the limitations of our study: duration of delivery room resuscitation efforts is not taken into consideration because no accurate data is noted in the charts. also, the temperature of the delivery room at the time of delivery is unknown and, just like in other institutions, these temperatures are known to be below the who-recommended 25°c (2). the limitations of the study are small sample size and a low number of infants receiving extensive resuscitation efforts in the delivery room. the effect of delivery room resuscitation procedures on admission temperatures in premature infants will require further investigation. promising results in maintaining normothermia in premature infants were recently published by seemedj 2018, vol 2, no. 1 impact of delivery room resuscitation efforts 34 southeastern european medical journal, 2018; 2(1) pinheiro et al. (18) regarding the use of chemical warming packs in achieving above-average admission temperatures in neonates. also, simple and inexpensive methods including preheating the delivery room in expectance of a premature birth have shown to reduce admission hypothermia rates (19). acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. bhatt dr, white r, martin g, van marter lj, finer n, goldsmith jp, et al. transitional hypothermia in preterm newborns. j perinatol 2007; 27 suppl 2:s45–47. 2. world health organization, maternal and newborn health/safe motherhood. thermal protection of the newborn: a practical guide. geneva, switzerland:world health organization; 1997. 3. lyu y, shah ps, ye xy, warre r, piedboeuf b, deshpandey a, dunn m, lee sk; canadian neonatal network. association between admission temperature and mortality and major morbidity in preterm infants born at fewer than 33 weeks' gestation. jama pediatr 2015;169(4):e150277. 4. laptook ar, salhab w, bhaskar b. admission temperature of low birth weight infants: predictors and associated morbidities. pediatrics 2007;119: e643–649. 5. harms k, herting e, kronm, schill m, schiffmann h. importance of preand perinatal risk factors in respiratory distress syndrome of premature infants: a logical regression analysis of 1100 cases. z geburtshilfe neonatol 1997;201(6):258-262 6. audeh s, smolkin t, bental y, haramati z, blazer s, litig e, biton r, dolberg s, makhoul ir. does admission hypothermia predispose to intraventricular hemorrhage in very-lowbirth-weight infants? neonatology 2011;100(4):373-9. 7. perlman jm, wyllie j, kattwinkel j, atkins dl, chameides l, goldsmith jp, et al. part 11: neonatal resuscitation: 2010 international consensus on cardiopulmonary resuscitation and emergency cardiovascular care science with treatment recommendations. circulation 2010;122(16 suppl 2):s516-38. 8. fawcett k. preventing admission hypothermia in very low birth weight neonates. neonatal netw 2014;33(3):143-9. 9. miller ss, lee hc, gould jb. hypothermia in very low birth weight infants: distribution, risk factors and outcomes. j perinatol 2011;31(suppl 1): s49-s56. 10. recommendations on the application of surfactant in management of idiopathic neonatal respiratory distress syndrome; section for neonatology and neonatal intensive medicine of the croatian society of perinatal medicine; gynaecol perinatol 2009;18(3):160–161. 11. papile la, munsick-bruno g, schaefer a. relationship of cerebral intraventricular hemorrhage and early childhood neurologic handicaps. j pediatr 1983;103: 273–7. 12. bell mj, ternberg jl, feigin rd, keating jp, marshall r, barton l, et al. neonatal necrotizing enterocolitis. therapeutic decisions based upon clinical staging. ann surg 1978;187: 1–7. 13. ricci z, ronco c. neonatal rifle. nephrol dial transplant 2013;28(9):2211-4. 14. de almeida mf, guinsburg r, sancho ga, rosa ir, lamy zc, martinez fe, et al. hypothermia and early neonatal mortality in preterm infants. j pediatr 2014;164(2):271-5. 15. fransson al, karlsson h, nilsson k. temperature variation in newborn babies: importance of physical contact with the mother. arch dis child fetal neonatal ed 2005;90:f500–504. 16. rojas-reyes mx, morley cj, soll r: prophylactic versus selective use of surfactant in preventing morbidity and mortality in preterm infants. cochrane database syst rev 2012;3:cd000510. 17. gupta n, saini ss, murki s, kumar p, deorari a. continuous positive airway pressure in preterm neonates: an update of current evidence and implications for developing countries. indian pediatr 2015;52(4):319-28. seemedj 2018, vol 2, no. 1 impact of delivery room resuscitation efforts 35 southeastern european medical journal, 2018; 2(1) 18. pinheiro jm, boynton s, furdon sa, dugan r, reu-donlon c. use of chemical warming packs during delivery room resuscitation is associated with decreased rates of hypothermia in very low-birth-weight neonates. adv neonatal care 2011;11(5):35762. 19. duryea el, nelson db, wyckoff mh, grant en, tao w, sadana n, chalak lf, mcintire dd, leveno kj. the impact of ambient operating room temperature on neonatal and maternal hypothermia and associated morbidities: a randomized controlled trial. am j obstet gynecol 2016;214(4):505.e1-7. seemedj 2017, vol 1, no. 2 risky behavior and exposure to noise among adolescents 27 southeastern european medical journal, 2017; 1(2) risky behavior and exposure to noise among adolescents 1 jelena tomac jovanović1, sabina cviljević2, božica lovrić2, tihomir jovanović2 1 high school pakrac, bolnička 59, croatia 2 general hospital požega, osječka 107, croatia corresponding author: sabina cviljević – sabina.cviljevic@po.t-com.hr introduction noise is an unwanted, i.e. unpleasant or unexpected sound; a mixture of sounds having various properties which can be permanent, received: july 20, 2017; revised version accepted: november 24, 2017; published: november 24. 2017 keywords: adolescents; hearing loss; noise abrupt and striking. the properties can vary in level, duration and distribution and can have multiple adverse effects on human health and hearing. noise is an audible acoustic energy which can negatively affect the physiological conclusion. study results showed that agnor count and structure cannot help in determining more clearly the border between parathyroid adenoma and hyperplasia in cytologic smears. (fustar preradovic lj, danic d, kardum-skelin i, sarcevic b, danic hadzibegovic a. agnor counts in differential diagnosis of parathyroid adenoma and hyperplasia in preoperative cytologic smears. seemedj 2017; 1(2); 19-26) abstract aim: adolescents are under greater risk of noise influence which affects their psychophysical health. the most common noise sources are too loud cinema halls, concerts, sports events, different outdoor events, street noise, and listening to music that is too loud on ipod and mp3 gadgets. the aim of this study was to investigate risk behaviors and attitudes among adolescents toward noise exposure; to explore how adolescents recognize symptoms and signs of noise influence to their own psychophysical health. methods: the study included 533 high school students aged 15-19 years. the data were collected using a questionnaire that contained a total of 32 questions/ attitudes/ statements. spss statistical software was used for data processing. results: students in higher grades (third and fourth) show a better understanding of noise types that can damage hearing and they agree with the statement that listening to loud music can damage hearing, whereas students of first and second grades disagree. younger students usually associate hearing loss with aging. conclusion: the study showed that such risky behavior of adolescents is in accordance with global trends. it can be and it has to be influenced by integrated programs within primary and secondary education. by continuous education of both children and parents when it comes to noise level in their living, working or entertainment environment, and when it comes to the ways in which they can decrease the noise level, damage to hearing can be prevented, or at least postponed. (tomac jovanović j, cviljević s, lovrić b, jovanović t. risky behavior and exposure to noise among adolescents. seemedj 2017; 1(2); 27-32) seemedj 2017, vol 1, no. 2 risky behavior and exposure to noise among adolescents 28 southeastern european medical journal, 2017; 1(2) and psychological state of humans. people are exposed to noise on a daily basis, including environmental, community, public and domestic noise, all of which represent one of the main issues of human environment, especially in city areas (1). main noise sources in outdoor spaces are traffic, industry, construction and public activities, sports and leisure. noise usually found in indoor spaces includes servicing equipment, music equipment and home appliances. traffic is one of the main causes of noise. in cities, noise emitted by cars accounts for 80 % of noise in city areas. what is more, next to busy road junctions, the noise can reach up to 90 db (2). about 80 million of europeans live in areas where the level of noise exceeds recommended exposure limits. by reducing partial or total noise primarily in the public and work environment as well as the time of exposure, at least half of hearing damage cases could be prevented. despite the fact that noise is an integral part of nature, factors such as industrial development, population growth and density and increased number of roads and vehicles, especially in urban settings, have led to an increase of noise exposure representing a threat to human health (table 1). the impact of noise on human health can be direct, resulting in damage to the organ of hearing and balance. also, it can be indirect and can affect the nervous, vascular, digestive and endocrine system. direct noise results in partial and complete hearing loss, tinnitus, various speech disorders, problems related to communication and balance disorders such as unsteadiness while walking. nowadays, noise is the main cause of hearing impairment. prolonged exposure to noise, such as during the whole day, permanently damages our hearing. noise induced by traffic, noise we are exposed to in school and at the work place, loud music, and most of all, neglecting to pay attention to our hearing, leads to noise-induced hearing loss (nihl). indirect effects on health include neurovegetative reactions such as hypertension, endocrine disorders and other metabolic disorders, as well as exhaustion, mental reactions (irritability) and low performance. depending on their interests, upbringing, education and habits, adolescents are at higher risk of noise influencing their health and physical and mental development. sources of noise are mostly extremely loud sound levels in cinemas, concerts, sports events, restaurants, open air events, shopping malls, street noise and loud listening to music on headphones. music played on ipods, cds and mp3 players is one of the least known, but one of the most dangerous killers of hair cells. the volume of sound emitted by these devices sends music via headphones/ earphones directly to our ear so that we no longer hear people around us or the noise of cars and trams, which means that the sound is too loud and potentially life-threatening. more than 78% to 90% of young people and approximately 50% of adults listen to music through headphones/earphones. about 50% of young people use personal music players from 1 to 3 hours per day and a significant number of young people even longer than that (3). it has been proven that exposure to noise of up to 85 db during an 8-hour period causes hearing loss, while the level of sound volume emitted by ipods and mp3 players is much louder (usually more than 100 db). adolescents and young adults consciously expose themselves to very loud music, very often for several hours in succession. loud music at concerts and in clubs, emitted by audio devices, represents a potentially dangerous source of noise. the noise level recorded at rock concerts ranges from 120 to 140 db, while in restaurants, at weekends, the noise level exceeds 95 db. according to a group of authors, professional exposure in nightclubs considerably exceeds the recommended noise level. taking the weekly exposure to noise in nightclubs into account, a 5-hour stay in a typical club will expose the ear to noise equivalent to 98 db (4). extremely high and very dangerous noise levels can be found next to loudspeakers at big music events (e.g. open-air concerts). on the back of every event ticket, there is a sentence written in very small letters which warns the visitors that loud music can damage the hearing. hence, the use of hearing protection, such as earplugs, which decrease the noise level but do not affect the quality of sound, is recommended to concert visitors. two decades ago, a group of authors came to the conclusion that a temporary threshold shift of more than 10 db can be observed after 3 hours of listening to music on seemedj 2017, vol 1, no. 2 risky behavior and exposure to noise among adolescents 29 southeastern european medical journal, 2017; 1(2) medium noise levels via portable audio devices (5). the aim of this study was to explore risk behavior of adolescents with respect to noise and their understanding of consequences of auditory overload on their psychophysical health. material and methods the study included 533 students, aged between 15 and 19, attending “pakrac” high school during november and december in 2016. the data was collected using a survey questionnaire especially designed for the purposes of this research. the questionnaire was divided into four parts. the first part included general data, like gender, date of birth, field of study the students are enrolled in. employing a likert-type scale, other parts of the questionnaire referred to statements on attitude, risky behavior and symptoms. the questionnaire contained a total of 32 questions/attitudes/statements. the research was approved by the school ethics committee. furthermore, the study was performed in accord with the ethical principles of the declaration of helsinki. in a meeting of all form teachers of “pakrac” high school, the aim of the questionnaire and the instructions on how to fill it in, were briefly presented. the questionnaires were filled in during homeroom class. the whole process of conducting the survey questionnaire went on smoothly, with occasional questions regarding the meaning of particular questions/attitudes stated in the questionnaire. statistical analysis the kolmogorov-smirnov test was performed to test the data against normal distribution. the mean values of the continuous data are expressed by the median and interquartile range, while nominal indicators are shown by absolute and relative numbers. the differences between categorical variables were tested using the χ2 test. in order to determine the difference between two independent samples, the mann whitney test was employed, while for three and more samples the kruskal wallis test was performed. also, originally written database programs and the statistical software package spps (version 15.0) were used, using a significance level of α = 0.05. results the study involved 533 participants (students) aged between 15 to 19. the sample included 200 (37.5%) male and 333 (62.5%) female students. while analyzing the research results, the participants were divided into two categories, i.e. junior and senior participants. the term “junior” refers to firstand second-year high-school students, while the term “senior” involves participants in the third, fourth and fifth grade. according to the obtained results, a statistically significant difference can be observed in the following attitudes: senior high-school students (third and fourth grade) show a statistically significant higher level of knowledge about table 1. environmental sounds and the corresponding sound level (noise) in decibels environmental sounds sound exposure level (db) threshold of hearing 0 – 25 whisper 20 conversational speech 40 tv 55 apartment on a busy street, noise in a large business office 60 busy traffic, noise in a cafe, 70 car, hair dryer 70 metro, big crowd 80 truck, busy crossroads 90 train passing 95 chainsaw, drilling machine 100 air hammer 105 loud music 110 rock concert near the speaker, thunder 120 a singer whose volume has reached the maximum 130 threshold of pain 130 –140 takeoff and landing of a plane 140 space shuttle platform 180 seemedj 2017, vol 1, no. 2 risky behavior and exposure to noise among adolescents 30 southeastern european medical journal, 2017; 1(2) different types of noise that can damage the hearing (p<0,05). also, they agree on the statement that loud music damages hearing (p<0,05) unlike the first-year and second-year students. furthermore, senior participants believe that noise causes the highest damage during leisure time (p<0,001), which can result in anxiety, depression and high blood pressure (p<0,05). in contrast to their senior counterparts, junior students often connect hearing loss with older people. the distribution of participants with respect to risky behavior is shown in table 2. a statistically significant difference regarding risky behavior can also be observed in the senior population of high-school students, who stated to rarely listen to music using headphones/earphones for more than an hour a day (p<0,05). all other statements relative to risky behavior do not point to a statistical significance between the different age groups. the highest overlap can be observed in the statement that noise has a negative effect on the students’ ability to learn, while the statements that the participants experienced ear pain as a result of listening to music using headphones/earphones and that the noise emitted by church bells and electronic devices in their living area (e.g. television, radio, air conditioner, refrigerator, washing machine, computer) made them feel uncomfortable, displayed the lowest level of agreement. discussion a lot of research has been carried out so far on the influence of noise on human health, especially that of young people, due to increasing exposure to levels that can directly and indirectly affect the health and quality of life. however, studies show that exposure to daily noise levels still represents a great public health and social issue, which has resulted in increased preventative measures. preventative measures first included the adult population exposed to professional noise, while, at the same time, the number of children and adults with hearing loss is constantly on the increase. the results of this research point to the necessity to educate table 2. participants distribution according to risk behavior risk behavior number (%) of respondents i do not agree at all i mostly disagree i neither agree nor disagree mainly i agree i agree total i listen to music every day for more than 1h through the headset 139 (26.1) 76 (14.3) 100 (18.8) 73 (13.7) 144 (27.1) 532 (100) family members complain that i listen to music or tv too loudly 209 (39.4) 108 (20.3) 59 (11.1) 72 (13.6) 83 (15.6) 531 (100) once a week (or more) i go to a nightclub or cafe bar 104 (19.6) 59 (11.1) 95 (17.9) 94 (17.7) 179 (33.7) 531 (100) i adhere to warnings of my smartphone that the music is too loud 216 (40.7) 84 (15.8) 97 (18.3) 75 (14.1) 59 (11.1) 531 (100) i often go to concerts and similar events 104 (19.6) 116 (21.8) 157 (29.6) 89 (16.8) 65 (12.2) 531 (100) when i'm exposed to noise, i use protection in the form of earplugs or ear pads 416 (78.6) 61 (11.5) 24 (4,5) 16 (3) 12 (2.3) 529 (100) i often participate in activities where i am exposed to noise 81 (15.2) 116 (21.8) 183 (34.4) 102 (19.2) 50 (9.4) 532 (100) i live near some sources of noise 301 (56.6) 96 (18) 69 (13) 40 (7.5) 26 (4.9) 532 (100) seemedj 2017, vol 1, no. 2 risky behavior and exposure to noise among adolescents 31 southeastern european medical journal, 2017; 1(2) adolescents with the aim to reduce their risky behavior and to influence their attitude towards noise. out of the total number of respondents, 80% disagree with the statement that listening to loud music through headphones/earphones is connected with hearing impairment, which represents a worrying fact about the perception of noise among young people. a study was carried out among 1547 students of 13-19 years of age in swedish schools. a significant difference was identified between socioeconomic and age groups. in other words, the age group of 13-15 displayed a better attitude towards noise, if compared to the age group ranging from 16 to 19. older adolescents stated to better take care of their hearing than younger adolescents (6). the results of the study in question are compatible with the results of this research, where older participants (3rd, 4th and 5th graders) are better acquainted with different noise types, risky behavior (listening to loud music) and the possible adverse effects. on the other hand, risky behavior of younger participants (1st and 2nd grade) is displayed in the amount of time spent listening to music using headphones/earphones, which is more than one hour. the same group of respondents (78.6%) do not use any kind of ear protection, be it earmuffs or earplugs. the cause of such an attitude could be found in the fact that younger participants connect hearing loss with older people. a six-year study carried out in the usa showed that 12% of children aged 6 to 19 suffer from noise-induced hearing-threshold shifts (7). research of some authors shows that 1% of all school children suffer from some kind of hearing damage (8). several years of exposure to loud music suffices to cause damage to the inner ear. it is estimated that after 10 years of exposure to loud music via audio devices in clubs and concerts, approximately 10% of people will suffer from irreversible bilateral hearing loss at a frequency of 3 khz (9). during 2005, a group of researchers performed a study on hearing loss among young people caused by noise. due to the fact that a large number of young people expose themselves to loud music in their free time, the researchers assumed that young people are unaware that exposure to loud music can result in hearing loss. in their research, they used a questionnaire which consisted of 28 questions. they presented the questionnaire on the public web site of a television network with the aim to find out the attitude of young people towards general health issues, including hearing loss. only 8% of respondents recognized the problem of hearing loss as a great health issue. the majority of participants experienced tinnitus or temporary, reversible hearing loss after visiting concerts (61%) and clubs (43 %). only 14% stated to have used earplugs. still, it is encouraging that the majority of the respondents would have been willing to use ear protection if educated or warned by a doctor about possible permanent hearing impairment (10). despite the evidence on the adverse effects of noise on young people, neither guidelines nor safety standards have been defined yet. scientists warn that today’s generations of young people are at higher risk of facing hearing damage at an earlier age than older generations. if music is listened to too often, doctors recommend keeping the volume at 60% of the maximum. as many as 22.4% of our participants sometimes experience buzzing in the ears; however, we do not possess any information as to whether this percentage increases with age. today’s studies indicate that the number of tinnitus cases will rise with age in children due to long-term exposure to noise. 45.1% of our participants indicated to have concentration problems due to environmental noise, while 57.3% answered to have problems with learning when noise is present. furthermore, 66% of the students study the easiest and most effectively when in silent environments. the results confirm that the presence of noise makes young people feel uncomfortable and that it causes lack of concentration. hearing loss can be prevented if children and parents are adequately educated about noise levels in the environment they live, work or entertain themselves in and about the methods they can use to reduce noise. conclusion the research has shown that risky behavior in adolescents follows global trends. the trends can be altered by using integrated programs during primary and secondary school education. seemedj 2017, vol 1, no. 2 risky behavior and exposure to noise among adolescents 32 southeastern european medical journal, 2017; 1(2) also, it is very important to educate the educators, who will then better convey their knowledge in a way that is more appealing to young people. young people are more prone to change their habits, hence quality education can lead to easier changes in young people’s attitude towards noise. raising public awareness and promoting activities regarding the prevention of hearing loss will improve the health of the ear and hearing. some of the preventative activities include restriction on exposure to loud noise, reduction of noise levels whenever possible or avoidance of noise sources. it is of utmost importance to perform further research on risky behavior and exposure to noise in youth in order to publish quality literature adapted to the current situation. this way, we can achieve better results and education aimed at the core of the problem. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. gomzi m, svakodnevna buka i moguće zdravstvene posljedice; stručni skup „buka i zdravlje“, 08.prosinac, zagreb 2005., knjiga sažetaka, str. 5-7. 2. klančnik m, utjecaj buke na zdravlje i radnu sposobnost. javno zdravstvo – nastavni zavod za javno zdravstvo splitskodalmatinske županije 2013; 2:12-14. 3. rosandić m, bonetti l. izloženost mladih u hrvatskoj buci – navike, stavovi, svijest o riziku, uporaba zaštite i rane posljedice. logopedija 2014; 4 (1):31-34. 4. williams w, beach ef, gilliver m. clubbing: the cumulative effect of noise exposure from attendance at dance clubs and night clubs on whole-of-life noise exposure. noise health 2010; 12:155-8. 5. lee pc, senders cw, gantz bj, otto sr. transient sensorineural hearing loss after overuse of portable headphone cassette radios. otolaryngol head neck surg 1985;93 (5):622-5. 6. olsen widén se, erlandsson si. the influence of socio-economic status on adolescent attitude to social noise and hearing protection. noise health 2004;7(25):59–70. 7. niskar as, kieszak sm, holmes ae, esteban e, rubin c, brody dj. estimated prevalence of noise-induced hearing threshold shifts among children 6 to 19 years of age: the third national health and nutrition examination survey, 1988-1994, united states. pediatrics 2001; 108:40–43. 8. blair jc, hardegree d, benson pv. necessity and effectiveness of a hearing conservation program for elementary students. j educ audiol 1996; 4:12–16. 9. maassen m, babisch w, bachmann kd, ising h, lehnert g, plath p, plinkert p, rebentisch e, schuschke g, spreng m, stange g, struwe v, zenner hp. ear damage caused by leisure noise. noise health 2001; 4:1-16. 10. chung jh, des roches cm, meunier j, eavey rd. evaluation of noise-induced hearing loss in young people using a web-based survey technique. pediatrics 2005;115(4):861–867. seemedj 2017, vol 1, no. 1 vocal cord paralysis and parathyroid cyst 136 southeastern european medical journal, vol 1, 2017. vocal cord paralysis and parathyroid cyst 1 davorin đanić1, ljubica fuštar preradović2, rajko jović3, ana đanić hadžibegović1 1 department of ear, nose and throat, head and neck surgery, “dr josip benčević” general hospital, slavonski brod; department of ear, nose and throat and maxillofacial surgery, osijek school of medicine, josip juraj strossmayer university in osijek, osijek, croatia 2 pathology and cytology laboratory, dr josip benčević general hospital, slavonski brod, croatia 3 clinical department of ear, nose and throat, cinical center vojvodina, novi sad, serbia corresponding author: assist. prof. ana đanić hadžibegović, md, phd ana_djanic@yahoo.com received: april 27, 2017; revised version accepted: may 25, 2017; published: may 26. 2017 keywords: parathyroid lesion; cyst; vocal cord; paralysis; cytology abstract aim: parathyroid gland lesions are an extremely rare clinical entity, mostly manifesting as adenoma and hyperplasia, rarely as parathyroid cysts, and most infrequently as carcinoma. parathyroid cysts are frequently asymptomatic lesions of the neck and the superior mediastinum with only 300 cases reported in the literature. symptomatic parathyroid cysts are very rare. vocal cord paralysis due to recurrent laryngeal nerve dysfunction may herald the presence of neck and mediastinal disease including a variety of neoplastic, inflammatory and vascular conditions. the aim of this report is to describe their clinical presentation, diagnostic procedures, and therapeutic approaches. the objective of the study was to assess the presence of vocal cord paralysis and recurrent laryngeal nerve dysfunction, as well as their association in patients with recurrent laryngeal nerve. methods. we performed a 12-year departmental review of parathyroid lesions and parathyroid cysts. retrospective analysis of clinical data on 20630 unselected patients submitted to thyroid gland and neck ultrasonography yielded 149 (0.007%) patients with parathyroid lesions, six (0.0003%) of them with parathyroid cysts. a comprehensive review of the literature revealed 18 patients with vocal cord paralysis and parathyroid cysts. results: parathyroid adenoma were present in 97/149 (0.65%) and hyperplasia in 46/149 (0.30%) patients with parathyroid lesions. parathyroid cysts were present only in six of 149 (0.04%) patients with parathyroid lesions. five patients had asymptomatic nonfunctional parathyroid cysts, with vocal cord paralysis diagnosed in one female patient. in the 19 patients with parathyroid cysts and vocal cord paralysis reported in the literature (including the patient reported here), nine and ten patients had parathyroid cysts localized in the neck and mediastinum, respectively. mediastinal cysts were twofold greater than those localized on the neck, while left recurrent laryngeal nerve dysfunction was recorded in 80% of cases. cyst removal resulted in recurrent laryngeal nerve functional recovery in two-thirds of patients. seemedj 2017, vol 1, no. 1 vocal cord paralysis and parathyroid cyst 137 southeastern european medical journal, vol 1, 2017. introduction parathyroid glands and their lesions are present in four different histopathologic and clinical entities, mostly as adenoma and hyperplasia, and rarely as cysts or carcinoma. parathyroid cysts (ptcs) are an uncommon cause of vcp. ptcs are benign neck tumors and that account for 0.8%-3.41% of all parathyroid lesions (1, 2). the true prevalence of ptc remains uncertain, and more than 300 patients with cystic lesions of parathyroid glands have been reported in the literature. about 200 of these lesions were localized in the neck, and the rest occurred in the mediastinum (2, 3). ptcs mostly occur at the age of 40-50 with a female predominance (female to male ratio, 2.5:1) and a few cases in pediatric patients. generally, the left side of the neck and inferior parathyroid glands are involved. true ptcs, found in 80% of patients, are nonfunctional and asymptomatic. occasionally, depending on their size and localization, ptcs may cause dysphagia, pain, and tracheal compression or rln lesion (2, 3). functional ptcs that develop by parathyroid adenoma cystic degeneration are found in 20% of patients (3). these ptcs usually occur as solitary lesions, whereas multiple functional ptcs are extremely rare and are associated with hyperparathyroidism (2, 3, 4, 5). only one case of malignant ptc transformation has been described in the literature (6). the etiology of vocal cord paralysis (vcp) is very heterogeneous and the true incidence of vcp in the general population is hard to estimate. according to shafkat et al., 42 cases per 10 000 new patients can be expected at ent clinic (7). most frequently, vcp develops as a sequel of recurrent laryngeal nerve (rln) dysfunction caused by a malignant tumor, various inflammatory processes or vascular malformations. a malignant tumor localized in the neck or mediastinum leads to rln dysfunction by its aggressive growth, pressure or traction, and most frequently by nerve infiltration. damage to rln is considerably less commonly induced by benign tumors of the neck. the incidence of rare and idiopathic vcp varies from 16.3% to 31.1% (8, 9). current diagnostic tools including ultrasonography, multi-slice computed tomography (msct), magnetic resonance imaging (mri) and technetium-99m sestamibi scanning can often differentiate solid tumors from cystic lesions. however, differentiating a thyroid cyst from ptc is challenging. ultrasound-guided fine needle aspiration cytology (us-fnac) is a useful test to confirm the diagnosis in case of clinically suspected parathyroid glands and parathyroid lesions, but also to detect a parathyroid gland of an unexpected localization, e.g., in the thyroid bed or within the thyroid gland. a parathyroid lesion may present as thyroid incidentaloma, a lesion incidentally found within or adjacent to the thyroid gland (10) (fig. 1). the aim of the present study was to evaluate the clinical characteristics, diagnostic difficulty and our experience in therapy of ptcs. material and methods study design: a retrospective analysis was performed on a cohort of 20 630 patients who submitted to ultrasonography examination of conclusion: parathyroid cysts are rare, mostly asymptomatic pathologic lesions of parathyroid glands that should be taken into consideration in patients with hyperparathyroidism or vocal cord paresis. (đanić d, fuštar preradović lj., jović r, đanić hadžibegović a. vocal cord paralysis and parathyroid cyst, seemedj 2017;1(1);136-143) seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 138 southeastern european medical journal, vol 1, 2017. the thyroid gland and neck. thyroid gland and neck ultrasonography reports were reviewed for diameter and the location of the suspect parathyroid lesions. ultrasound-guided fine needle aspiration cytology (us-fnac) of the parathyroid gland was performed under continuous, ultrasound visualization with use of an acusion x 300 device using 8.9 mhz and 11 mhz probes. the medical records were reviewed for clinical symptoms, biochemical measurements, ultrasonography reports, cytology data, surgical treatments and pathology reports. statistical analysis proportions were expressed as percentages, whereas all continuous variables were calculated as median and range. there were no interor intra-group comparisons needing further statistical analyses. results the analysis yielded 149 patients with parathyroid lesions out of 20630 examinations (0.007%), including 97 (65.1%) patients with parathyroid adenoma, 46 (30.8%) patients with hyperplasia and six (4.0%) patients with ptcs. there were four female and two male ptc patients with a median age of 52.5 (minimum 44, maximum 79, range 35) years and a median cyst size 21.5 mm (minimum 17, maximum 45, range 28 mm) (table 1). these six patients with ptcs underwent usfnac, and the presence of parathyroid hormone (pth) was confirmed by aspirate biochemistry on a cobas e411 analyzer (roche, mannheim, germany). nonfunctional ptcs were present in five patients and vcp in one patient. one patient had a functional ptc, with a parathyroid hormone (pth) level of 77.61 pg/ml and normal serum and ionized calcium levels. the surgical extirpation of the cysts and histopathologic analysis were performed in three patients, whereas the other three patients underwent usfnac (fig. 2 and 3). the study protocol was approved by the hospital ethics committee and by the zagreb school of medicine ethics committee. a comprehensive review of the literature from 1953 to the present showed 19 patients with ptc and vcp, including our patient reported herewith (table 2). there were 53% males, with a median age of 54 (minimum 29, maximum 83, range 54) years, and a median cyst size of 48 mm (minimum 12, maximum 123, range 111 mm). ten table 1. clinical and laboratory characteristics of parathyroid cyst patients (n=6) patient no. initials, age (yrs)/gende r symptoms localization cyst size serum pth pg/ml cyst pth pg/ml therapy: fna/surger y 1 51/f osteodynia left inferior 21 mm 35.43 240.1 fna 2 76/f hoarseness left vocal cord paresis left inferior 22 mm 30.31 389.5 surgery 3 54/f dry cough nonspecific breathlessne ss at exertion left inferior 24 mm 29.22 498.0 surgery 4 44/f left-sided hemithyroid ectomy right inferior 17 mm 64.3 21 770 fna 5 50/m nephrolithia sis left superior 45 mm 77.61 103.1 fna 6 79/m nonspecific swallowing disturbance right inferior 18 mm 110.90 337.0 surgery pth = parathyroid hormone; fna = fine needle aspiration seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 139 southeastern european medical journal, vol 1, 2017. ptcs were localized in the mediastinum and nine in the neck; there were 16 nonfunctional ptcs. functional ptcs were present in three female patients, accompanied by elevated serum pth and calcium levels. left-sided vcps were found in 15 patients. in 80% of patients, ptcs were surgically removed in toto, whereas 20% of patients underwent transcutaneous aspiration of the cyst content. in two-thirds of patients, ptc removal resulted in functional rln recovery. discussion parathyroid lesions are rare clinical entities, and parathyroid cysts are uncommon forms of parathyroid lesions, possibly even referred to as 'a forgotten diagnosis' (27). microcysts of the parathyroid gland are a common occurrence in the normal population and are found primarily in aging glands along with increased fat content. macroscopic cysts of the parathyroid glands that are larger than 10 mm in diameter are referred to as parathyroid cysts and necessitate investigation. the clinical presentation is very different; functional parathyroid cysts are relatively easier to detect than nonfunctional ones. ptcs are diagnosed by clinical symptoms and biochemical parameters of hyperparathyroidism with elevated serum calcium levels, and postoperatively by histopathology demonstrating parathyroid gland tissue in the cyst wall and the postoperative correction of the pth level and table 2. reported 19 cases of vcp associated with benign parathyroid cysts reference public. date first author age/ gender local. size mm functional cyst status neck side treatment postop. status 11. 1953. crile g. jr 69/f m 60 nf r s n 8. 1966. hayashi s. 48/m m 83 nf r s n 12. 1978. clark o.h. 29/f m 63 nf l s n 13. 1983. marco v. 42/m m 12 nf l s n 14. 1988. takahashi e. 68/f m 73 f r s n 15. 1990. delauny t. 71/f m 40 f l s n 16. 1991. coates g. 60/m n 80 nf l a p 8. 1992. narita y. 54/m m 123 nf l s n 17. 1993. grey a.b. 38/f n 20 nf l a n 18. 1997. landau o. 77/m m 88 nf r s p 19. 2000. sen p. 37/f n 25 nf l a n 20. 2006. coelho d.h. 49/m n 48 nf r s n 21. 2006. sihoe a.d. 51/m m 95 nf l s n 22. 2008. woo e.k. 83/m n 39 nf l s p 23. 2011. ghervian c. 50/f n 42 nf l a n 24. 2012. khan a. 58/f n 32 f r s p 25. 2013. menezes vc 56/m m 72 nf l s n 26. 2015. arduc a. 30/m n 30 nf l s n 2016. danic d. 76/f n 22 nf l s p nneck; m-mediastinum; ffunctional; nfnonfunctional, ssurgery; aaspiration; pparalysis rln; nnormal functional status rln seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 140 southeastern european medical journal, vol 1, 2017. hypercalcemia (3). generally nonfunctional parathyroid cysts are asymptomatic and incidentally diagnosed during clinical work-up for thyroid disorders or other neck and chest soft tissue tumors (10, 27). vocal cord paresis can be caused by recurrent laryngeal nerve lesions of various etiologies. surgical traumas and tumors are most commonly involved, tough the cause remains unknown in one-third of lesions. the rare causes of vcp include cardiovascular syndromes, cervical spine osteophytes, tracheal diverticula, subclavian artery aneurysm, jugular vein thrombosis, and meningomyelocele (9). ptc has been described in several studies as a rare cause of vcp. ho and mcmullen report on 13 patients with various parathyroid gland lesions considered to have caused vcp. functional ptcs were present in six patients, four of them with the clinical picture of hyperparathyroidism and a histopathology report indicating adenoma, and two patients with secondary hyperparathyroidism and a histopathology report indicating hyperplasia. three patients had both ptcs and parathyroid gland nonfunctional hyperplasia, whereas five patients had true nonfunctional ptcs, two of them suffering from vcp and clinically present tumor mass on the neck, and one patient complaining of pain in the neck (27). ptcs belong to a group of extremely rare causes of vcp. in their study that included 93 patients with mediastinal ptcs, shields and immerman recorded nine (9.5%) patients with vcp. true nonfunctional ptcs were found in seven patients and functional ptcs in two female patients (5). in our unselected cohort of 20 630 patients that underwent neck and thyroid gland us, parathyroid lesions were recorded in 147 (0.007%) patients and ptcs in only six (0.0003%) patients, with vcp found in only one female patient (table 1). that patient was a 76-year-old female presented for clinical ent examination due to hoarseness persisting for months. fiberoscopy, laryngostroboscopy and electromyography studies revealed left-sided vcp. standard biochemistry parameters showed no pathologic findings. the patient underwent neck and thyroid us examination (figure 1). based on these findings, the diagnosis of nonfunctional ptc was made. she underwent a minimally invasive surgical procedure. the cyst was figure 1. thyroid ultrasound showing cystic mass adjacent to the left lobe of the thyroid gland figure 2. thyroid preparation with adjacent cystic of a parathyroid gland (own source) figure 3. parathyroid gland histology (own source) histopathology: inner cyst surface lined with a layer of flat epithelial cells; parathyroid gland tissue along the cyst connective wall (hemalaun-eozin stain) seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 141 southeastern european medical journal, vol 1, 2017. surgically extirpated in toto, while saving rln carefully (figure 2). follow up biochemistry parameters obtained 24 h postoperatively were within reference range. histologic examination confirmed the presence of ptc (figure 3). at 12 months after the surgery, the patient had leftsided vcp and dysphonia, without breathing and swallowing difficulties. serum levels of pth, calcium and phosphorus were within the reference range. about 300 patients with ptcs have been reported in the literature to date, 200 of these localized in the neck and 100 in the mediastinum (1, 5, 27). analysis of the available literature yielded 19 patients with ptc and vcp, including our patient reported herewith (table 2). the number of patients with neck and mediastinal ptcs was almost equal, i.e., nine and ten, respectively. a review of the present literature shows the prevalence of vcp was twofold greater (10 %) in patients with the ptc localized in the mediastinum than in those with the ptc in the neck (4.5 %). this finding may be due to the twofold greater median cyst diameter (75.5 mm) and probably a higher compression of the cyst wall upon rln in patients with mediastinal ptcs as compared with the median neck cyst size of 37.5 mm. in our patients, the median ptc size was significantly lower, i.e., 24.6 mm. in 80% of patients, ptcs were localized on the left side of the neck or mediastinum with left-sided vcp. left rln is known to be more vulnerable than right rln because of its length and localization. in two-thirds of patients, ptc removal resulted in the functional recovery of rln. a review of the present 19 patients according to the functional cyst status shows that 16 patients had asymptomatic ptcs and three female patients had functional ptcs. loss of rln function can be caused by a variety of mechanisms. in some patients, loss of rln function can be explained by the known and verified pathologic mechanisms. a large cystic growth exerts nerve compression and/or traction, while local inflammation or edema can lead to neurapraxia and eventually to axonotmesis with a loss of function. in patients with long-standing renal insufficiency, the loss of rln function can occur as a sequel of a hemorrhage into the ptc, frequently associated with inflammation (27). major intracystic hemorrhages and rln lesions were found in patients with hypercalcemic episodes and functional ptcs (24). in the majority of these patients, nerve and vcp recovery ensued after ptc removal. in malignant tumors, of the thyroid gland in particular, nerve lesions can be induced by direct invasion of malignant cells or by nerve compression by nodular hyperplasia in the case of a benign growth. however, in some cases, the cause of the loss of rln function remains obscure, as in our patient. accordingly, in some ptc patients, the loss of rln function and vcp appear to occur due to some other unidentified cause, or vcp and ptc are present concurrently but without mutual interaction. the modality and extent of ptc treatment is determined by its size, localization, functional status, and cyst-related complications. minimally, invasive surgery for ptc removal is the gold standard in the treatment of functional ptcs. in patients with true nonfunctional ptcs and symptoms of adjacent organ compression, we believe that surgical removal of the cystic lesion is necessary, along with relieving the neck or mediastinum structure compression, with special attention paid to rln. percutaneous cyst aspiration is a satisfactory therapeutic procedure in small, asymptomatic, nonfunctional and uncomplicated cysts (27). in patients with permanent vcp, speech therapy, augmentation injections, or unilateral medialization laryngoplasty should be used. in our patient, three months of speech therapy resulted in satisfactory voice quality. there were several limitations of our study. the most important ones were retrospective design, a single center experience, and a small number of cases that did not allow any statistical analyses. in conclusion, our study has demonstrated that parathyroid cysts are rarely encountered in general neck and mediastinum pathology, but should not be forgotten as a possible cause of unilateral vcp. functional and larger ptcs are easier to diagnose than small and asymptomatic ones. us-fnac and aspirate biochemistry for seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 142 southeastern european medical journal, vol 1, 2017. pth are the standard diagnostic procedures for this entity. minimally invasive surgery with in toto cyst extirpation is the gold standard in the treatment of all cystic lesions associated with complications. funding: no specific was received for this study. competing interests: none to declare. references 1. pontikides n, karras s, kaprara a,cheva a, doumas a, bostsios d, oschidis a, efthimiou e, wass j, krassas g. diagnostic and therapeutic review of cystic parathyroid lesions. hormones 2012;11(4):410-8. 2. da silva da, machado mc, brito ll, guimaraes ra, fava as, mamone mcac.parathyroid cysts: diagnosis and treatment. rev bras otorhinolaringol 2004;70 (6):840-4. 3. wirowski d, wicke c, bohner h, lammers bj, pohl p, schwarz k, goretzki pe. presentation of 6 cases with parathyroid cysts and discussion of the literature. exp clin endocrinol diabetes 2008;116(8):501-6. 4. entwistle jw, pierce cv, johnson de, o'donovan sc, bagwell ce, salzberg am. parathyroid cysts: report of the sixth and youngest pediatric case. j pediatr surg 1994;29:1528-9. 5. shields tw, immerman sc. mediastinal parathyroid cysts revisited. ann thorac surg 1999;67:581-90. 6. vazquez fj, aparicio ls, gallo cg, diehl m. parathyroid carcinoma presenting as a gigant mediastinal retrotracheal functional cyst. singapore med j 2007;48:e 304. 7. shafkat a, muzamil a, lateef m. a study of incidence and etiopathology of vocal cord paralysis. indian j otolaryngol head neck surg 2000;54:30-2. 8. kay-rivers e, mitmaker e, payne rj, hier mp, mlynarek am, young j, forest vi. preoperative vocal cord paralysis and its association with malignant thyroid disease and other pathological features. j otolaryngol head neck surg 2015;44:35. 9. sobrino-guijarro b, virk js, singh a, lingam rk. uncommon and rare causes of vocal fold paralysis detected via imaging. j laryngol otol 2013;127:691-8. 10. fuštar-preradović lj, šarčević b, đanić d. morphometry in differential diagnosis of pathologically altered parathyroid glands: adenoma and hyperplasia. coll antropol 2012;36:47-1. 11. crile g jr, perryman rg. parathyroid cysts. report of five cases. surgery 1953;14:151-4. 12. clark oh. parathyroid cysts. am j surg 1978;135:395-402. 13. marco v, carrasco ma, marco c, bauza a. cytomorphology of a mediastinal parathyroid cyst. report of a case mimicking malignancy. acta cytol 1983;27:688-92. 14. takahashi e, kohno a, narimatsu a, miyake h, kimura f, shigeta a, itaoka t, wada y. a case of mediastinal parathyroid cyst. jpn j clin radiol 1988;33:393-6. 15. delauny t, peillon c, manourvrier jl, deotto jf, doucet j, nicaise jm, watelet j, testart j. cysts of the parathyroid glands. apropos of 6 cases ann chir 1990;44:231-5. 16. coates g, pearman k, holl-allen rtj. recurrent nerve palsy duo to parathyroid cyst. int surg 1991;76:192-3. 17. grey ab, shaw jh, anderson ne, holdaway im. parathyroid cyst with recurrent vocal cord paresis. aust n z j surg 1993;63:561-2. 18. landau o, chamberlain dw, kennedy rs, pearson fg keshavjee s. mediastinal parathyroid cysts. ann thorac surg 1997;63:951-3. 19. sen p, flower n, papesch m, davis a, spedding av. a benign parathyroid cyst presenting with hoarse voice.. j laryngol otol 2000;114:147-8. 20. coelho dh, boey hp. benign parathyroid cyst causing vocal cord paralysis: a case report and review of the literature. head neck 2006;28:564-6. 21. sihoe ad, chir b, cheng lc, sit ak. mediastinal parathyroid cyst: a rare benign cause of voice hoarseness. chest 2006; 130 (4_meeting abstracts).347b-b 22. woo ek, simo b, conn b, connor sej. vocal cord paralysis secondary to a benign parathyroid cyst: a case report with clinical, imaging and pathological findings. eur radiol 2008;18:2015-8. 23. ghervan c, goel p. parathyroid cyst, a rare 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seemedj 2018, vol 2, no. 2 emotional control original article emotional control in surgical and intensive care nursing: sociodemographic differences 1 stana pačarić 1,2, ana nemčić 1, nikolina farčić 1,2, vanja trazer 1 1 university hospital centre osijek, osijek, croatia, department of surgery 2 faculty of medicine osijek, university of osijek, osijek, croatia corresponding author: nikolina farčić, nikolina.farcic@mefos.hr received: jan 11, 2018; revised version accepted: feb 22, 2019; published: mar 31, 2019 keywords: emotional intelligence, self-control, nurses, thinking abstract aim: to determine whether there is a difference in the impact of emotion on memory, behaviour, thinking and mood with regard to age, gender, level of education and length of service. methods: research included 105 nurses. it was conducted anonymously by a standardized emotional regulation and control questionnaire (erik). results: average rating on the scale was somewhat lower for men, respondents under 30 years of age and respondents with a university degree. average rating on the emotional regulation and control scale were significantly higher for respondents with 31 and more years of service (kruskalwallis test, p=0.046). regarding male respondents, there is a significant correlation of age e on the scale. regarding female correspondents, there is no significant correlation between age and their overall score on the emotional regulation and control scale and sub-scales. considering the age of respondents, results indicate that the decrease in the ability to control emotional reactions is proportional to the increase in age, but not to a significant degree. regarding elderly respondents, the value of emotional regulation and control is higher in comparison to younger respondents (spearman's cor conclusion: increase in length of service decreases the ability to control emotional reactions and there are no significant differences in emotional control with regard to gender, age and educational background. (pačarić s, nemčić a, farčić n, trazer v. emotional control in surgical and intensive care nursing: sociodemographic differences. seemedj 2018; 2(2); 1-7) seemedj 2018, vol 2, no. 2 emotional control introduction emotional intelligence is defined as the ability to monitor one's own and other's emotions and as the ability to perceive, assess and express emotional information by creating feelings for emotional and intellectual development (1). as such, it has an indirect role between mental health and stress. people with a higher level of emotional intelligence have a greater ability to deal with conflicts and the environment than people with a lower level of emotional intelligence (2). emotional intelligence is composed of five main elements: selfawareness, self-regulation, internal motivation, empathy and social skills. these five elements of emotional competence have been defined as levels built one on another, which ultimately represent emotional intelligence as a whole (3). pence (4) points out that emotional intelligence can help nurses in managing their own and patient’s emotions; it reflects a real emotional reaction, where feelings and communication are exchanged during nurse-patient interaction. nursing is considered an emotionally demanding job because nurses are required to cope with various emotional demands and affective behaviours in their work-related relationships, be it with their patients, relatives or colleagues (5). emotions play an important role in the nursing practice and in order to understand and manage them, they first must be properly identified. even though this sounds simple, it is often rather difficult to accomplish. nurses can desire to express their emotional states, yet they should minimize their display. this can make them appear inauthentic and thus produce a negative effect on both the provision of optimal nursing care and the nurse-patient relationship (6). the problem of work burnout can cause emotional stress, emotional burnout, depersonalization, feeling of failure, stressrelated illnesses, demotivation and dissatisfaction with care, reduced quality of care and conflicts with other team members and patients (7). putting on one’s professional face highlights the need of nurses to use a “mask” to control their emotional states and to maintain their professional persona whilst caring for patients (8). in the last decade, there has been an increase in the interest for the implementation of programs aimed at developing social and emotional skills, such as problem-solving, decision-making, communication skills, recognition of own and others' emotions, control of emotions, control of one’s own behaviour and so on (9). emotional regulation relates to the ability of managing one’s own emotions in situations when particular emotions are unfavourable, occur at inconvenient times or when their intensity is inappropriate (10). emotional regulation relates to heterogeneous processes that regulate which emotions we feel, when we feel them and how we experience and express them. there is a consensus among researchers that emotions have more components in an experiential, behavioural and physiological domain. emotional regulation involves a change in one or more of these systems and does not need to (but it certainly can) include attempts to change the subjective experience of emotions (11). research has shown that the ability of emotional self-regulation can help in coping with emotional demands of jobs (12). emotional regulation is a new research area that provides nurses with an opportunity to explore their value and become more efficient in managing stressful situations in the working environment and improving the full range of nursing practice (13). objectives of this research are to determine whether there is a difference in the impact of emotion on memory, behaviour, thinking and mood with regard to age, gender, level of education and length of service. respondents and methods research subjects were 105 nurses employed at the department of surgery in intensive care units and operating rooms at the clinical hospital osijek. of 105 respondents, 29 (28%) were men and 76 (72%) were women. most respondents – 79 (76.7%) of them – had secondary school qualifications. the mean age of respondents was 38 years, whereas the average length of service was 16.5 years. seemedj 2018, vol 2, no. 2 emotional control research was conducted during may and june 2015 and it included nurses holding the position of operating nurse in the operating room and the position of intensive care nurse at the department of surgery of the clinical hospital osijek. the respondents were informed about emotional assessment and control in nursing in both written and oral form. they read the form/questionnaire related to the research and were introduced to the research process. introduction to research included a presentation of basic information about the research, main objective, manner of carrying out the research, confidentiality and data protection. the survey was voluntary and anonymous. the ethics committee of the clinical hospital osijek granted its consent to conduct the research. completed forms/questionnaires were submitted personally in an envelope in order to ensure anonymity. a standardized emotional regulation and control questionnaire was used upon permission from the author vladimir takšić. the questionnaire assessed the impact of unpleasant emotions and mood on thinking, memory and behaviour of individuals. the questionnaire consisted of 20 items that assessed the size of the effects of negative emotions and moods on thinking, memory and behaviour of the individual and on the ability of emotional control (takšić, v.: emotional regulation and control scale, erik, 2003) (14). the subjects rated 20 statements about emotional regulation and control from 1 to 5 on the likert scale, where 1 was (not at all), 2 (seldom), 3 (occasionally), 4 (mostly) and 5 (completely). the first part consisted of statements related to the effects of emotions and mood on thinking and behaviour (8 statements), followed by statements related to the memory of emotionally saturated contents (6 statements) and to statements related to control of their own emotional reactions (6 statements). higher scores indicated poorer emotional regulation and control. the second part of the questionnaire contained general information about sociodemographic characteristics (age, gender, length of service, qualifications). statistical methods the figures were described by median and interquartile range. categorical variables were described in absolute and relative frequencies. fisher's exact test and mann-whitney test were used to determine the differences between independent groups (age, gender, education). the level of significance was set at α = 0.05. the analysis of the data obtained was done by a programming system spps for windows (version 13.0). results the study included 105 respondents, of which 29 (28%) were men and 76 (72%) were women. the respondents’ ages ranged from 24 to 64, their mean age was 38 (an interquartile range of 3048 years) and 5 (4.9%) respondents had a university degree. average length of service was 16.5 years (an interquartile range of 8-26 years), ranging from 1.5 to 45 years of service, with no significant differences in gender (table 1). seemedj 2018, vol 2, no. 2 emotional control table 1. respondents by characteristics and gender men women total p total 29 (28%) 76 (72%) 105 (100%) age* 40 (28 – 49.5) 38 (31.5 – 47) 38 (30 – 48) 0.985† length of service* 19 (7.5 – 28) 16 (8.5 – 25) 16.5 (8 – 26) 0.894† level of education high school degree 24 (82.8%) 55 (74.3%) 79 (76.7%) 0.770‡ higher education 4 (13.8%) 15 (20.3%) 19 (18.4%) university degree 1 (3.4%) 4 (5.4%) 5 (4.9%) age – 30 years 10 (34.5) 16 (21.1) 26 (24.8) 0.348‡ 31 – 40 6 (20.7) 27 (35.5) 33 (31.4) 41 – 50 8 (20.7) 23 (30.3) 31 (29.5) 51 years and above 5 (17.2) 10 (13.2) 15 (14.3) length of service – by groups up to 10 years 11 (37.9) 20 (26.3) 31 (29.5) 0.286‡ 11 – 20 5 (17.2) 26 (34.2) 31 (29.5) 21 – 30 8 (27.6) 22 (28.9) 30 (28.6) 31 and above 5 (17.2) 8 (10.5) 13 (12.4) *median (interquartile range); †mann average rating on the scale was somewhat lower for men, respondents under 30 years of age and respondents with a university degree. however, the differences with regard to gender, age and educational background were not that significant. average ratings on the emotional regulation and control scale were significantly higher respondents with the length of service of 31 years or more (kruskal wallis test, p = 0.046) (table 2). table 2. average ratings on the scales and subscales in the emotional regulation and control questionnaire by gender, age, level of education and length of service* median (25% 75%) the impact of emotions and mood on thinking the impact of emotions and mood on memory control of emotional reactions the whole scale gender men 2.5 (2.2–2.9) 2.7 (2.4–3.3) 2.5 (2.2–3.1) 2.6 (2.4–3.2) women 2.7 (2.3–3) 3 (2.5–3.3) 2.3 (2–2.8) 2.7 (2.4–3.0) p value* 0.860 0.560 0.138 0.649 age – 30 years 2.4 (2.2-2.9) 3 (2.7-3.4) 2.2 (2-2.5) 2.5 (2.3-2.9) 31 – 40 2.5 (2.1-2.9) 3 (2.7-3.3) 2.3 (2-3) 2.7 (2.3-3) 41 – 50 2.8 (2.4-3.3) 2.8 (2.5-3.3) 2.5 (2-3) 2.8 (2.5-3.1) 51 and above 2.4 (2.1-3.4) 2.8 (2.3-3.3) 2.7 (2.3-3.3) 2.7 (2.4-3.4) p value† 0.308 0.838 0.220 0.440 level of education high school degree 2.6 (2.3-3) 3 (2.6-3.3) 2.3 (2.2-3) 2.7 (2.3–3) higher education 2.6 (2.1-3) 3.2 (2.7-3.3) 2.3 (2-2.8) 2.6 (2.3–3.1) university degree 2.3 (1.9-3.1) 2.5 (2.1-3.3) 2.2 (1.8-3.1) 2.2 (1.9–3.2) p value † 0.695 0.344 0.655 0.525 length of service up to10 years 2.5 (2.1–2.9) 3 (2.7–3.3) 2.2 (2–2.5) 2.5 (2.3–2.8) 11 – 20 2.6 (2.3–3.1) 3.2 (2.7–3.5) 2.5 (2.2–3.2) 2.8 (2.4–3.1) 21 – 30 2.8 (2.4–3.2) 2.9 (2.3–3.3) 2.5 (1.9–3) 2.7 (2.4–3.1) 31 years and above 2.4 (2.2–3.3) 2.7 (2.4–3.3) 2.7 (2.3–3.3) 2.6 (2.4–3.4) p value† 0.332 0.539 0.046 0.184 *mann-whitney test; †kruskal-wallis test seemedj 2018, vol 2, no. 2 emotional control in the group of men, there is a significant correlation between age (spearman's length of service (spearman's correlation score on the scale. in comparison to younger respondents, older respondents agreed mostly or entirely with the statements. regarding elderly respondents, control of emotional reactions was rated higher when compared to the ratings given by younger respondents 0.017). regarding female correspondents, there was no significant correlation between age and their overall score on the emotional regulation and control scale and sub-scales. discussion mood affects work-effectiveness of nurses (15). some authors have stated in their studies that people with a low level of positive emotions suffer from emotional exhaustion, which is one of the elements of burnout and the development of psychosomatic disorders and physical illnesses (16). nurses are usually asked to provide care in an unemotional manner, without demonstrating any emotional response to the pain; they have to be able to buffer their emotions, and to focus on medical care (17). if we consider the impact of emotions and mood on thinking and memory by gender, the results indicate that said impact received a higher average grade on the likert scale from women than from men. this suggests that emotions have a greater impact on opinion and memory in women than in men, although those differences are not significant. men and women use different sides of their brains to process and store longterm memories (18). the results also indicate that emotions have a greater impact on memory in women, in the sense that women remember negative emotions more than men do. these differences may be attributed to various genetic, hormonal and environmental factors. both sexes are equal in intelligence, but tend to operate differently. men and women appear to use different parts of the brain to encode memories, sense emotions, recognize faces, solve certain problems and make decisions (19). if we consider the age of respondents, there was no significant difference in the impact of emotions and mood on memory. however, it can be said that increase in age decreases the impact of emotions and mood on memory among nurses. there is a significant correlation between age and length of service, which is understandable because nurses who have been working longer are older. results indicate that the decrease in the ability to control emotional reactions is proportional to the increase in age, but not to a significant degree. regarding educational qualifications, the results indicate that the higher the level of education, the lower the impact of emotions and mood on thinking and memory. this means that people who are more educated can control emotional reactions better, but the differences are also not that significant. we know that emotional regulation is a complex construct which is from an educational perspective seen as an ability to use appropriate strategies in a particular context. therefore, it is presumed that emotional control can be learned and that education and practice must be combined (20). through the selfawareness process, nursing students can become aware of the importance of taking care of their own emotional health. furthermore, they can also learn how to effectively cope with complex interactions with their patients and colleagues (20). emotional regulation or control of allows people to comfort themselves and relieve anxiety, depression or irritability (21). emotional regulation includes managing of unpleasant emotions and emotional reactions, analysis of the causes of such emotions, choice of reaction and the ability to postpone fulfilment of a particular desire or need. in that sense, it determines the external behaviour of individuals and internal well-being (22). some scholars have discovered that emotional self-regulation can help nurses in recognizing and responding to patients’ emotions in an empathic manner, which enables them to provide better nursing care (23). however, as the number of years of service in the nursing profession increases, the level of nurses’ empathy decreases (24), which is also true for people working in the same department for a long period of time. results of a study in tehran have shown that length of seemedj 2018, vol 2, no. 2 emotional control service is related to continuance commitment and occupational commitment (25). when it comes to the impact of emotions and mood on the control of emotional reactions, the majority of respondents rated their emotional control on the likert scale as average. when people are in a good mood, they evaluate life in general as successful and fulfilled and they are more likely to remember positive events in their lives. in contrast, when they are in a bad mood, then such mood also affects their motivation, behaviour and everyday interactions (26). people in a good mood are nicer to other people, more generous and more willing to help. they are also more open to new activities and ideas and more creative than people in a bad mood (27). healthcare workers who are more competent in recognizing emotions and needs of patients are much more successful in taking care of themselves (28). average ratings on the scale were somewhat lower for men, respondents under 30 years of age and respondents with a university degree. however, the differences with regard to gender, age and educational background are not that significant. conclusion average ratings on the emotional regulation and control scale were significantly higher for respondents with 31 or more years of service. it has also been discovered that nurses with the shortest length of service are the least active in coping with problems, as well as that their competence increases with length of service (29). taking into consideration the demands of the nursing profession, it can be concluded that the possession of emotional intelligence and control is an imperative for successful professional activities. nurses should attend training programs that also include communication skills programs. it is also necessary to help nursing staff in adopting constructive emotional regulation strategies so that they could manage their and others’ emotions and apply such strategies in their work. nurses with better emotional regulation skills are more motivated to work and to take care of their well-being at home in situations when they have to face emotionally demanding situations at work, which indicates a spillover effect (30). acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare references 1. salovey p, mayer jd. emotional intelligence. imagin cogn pers 1990; 9(3): 185-211. https://doi.org/10.2190/dugg-p24e-52wk-6cdg. 2. augusto landa jm, lópez-zafra e, berrios martos m p, aguilar-luzón md. the relationship between 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[article in polish], https://www.ncbi.nlm.nih.gov/pubmed/20349619. 30. blanco-donoso lm, demerouti e, hernández eg, moreno-jiménez b, cobo ic. positive benefits from caring on nurses' motivation and well-being: a diary study about the role of emotional regulation abilities at work. int j nurs stud 2015; 52(4): 804-16. http://dx.doi.org/10.1016/j.ijnurstu.2015.01.002. http://dx.doi.org/10.1016/j.ijnurstu.2015.01.002 seemedj 2019, vol 3, no. 1 nutrition patterns in alzheimer’s disease 42 southeastern european medical journal, 2019; 3(1) review article nutrition patterns in prevention and treatment of neurodegenerative diseases: alzheimer’s disease 1 vesna šeper 1, nebojša nešić 1 1 college of applied sciences “lavoslav ružička” in vukovar, croatia corresponding author: vesna šeper, vseper@vevu.hr received: march 26, 2019; revised version accepted: may 16, 2019; published: may 31, 2019 keywords: alzheimer’s disease, inflammation, amyloid plaques, berries, supplements abstract background: the population of the elderly is increasing, as is the number of people suffering from neurodegenerative diseases. since the cause of those diseases is unknown, there is no appropriate medical treatment. the purpose of this systematic review is to present papers on consumption of certain foods, supplements or introduction of dietary restrictions that promote healthy brain aging or possibly delay the onset of disease. methods: the pubmed, sciencedirect, and springerlink databases were used for the research. 24 studies with a total number of 10 445 participants were selected as satisfying the final criteria. results: mediterranean diet, dietary supplements and natural nutrients with recently discovered pharmacological properties are of interest to numerous scientists. conclusion: since the results of the studies are inconsistent, we concluded that a large, carefully controlled long-term interventional study would be required in order to investigate the effect of nutrition on prevention of disease and cognitive decline. (šeper v, nešić n. nutrition patterns in prevention and treatment of neurodegenerative diseases: alzheimer’s disease. seemedj 2019; 3(1); 42-49) seemedj 2019, vol 3, no. 1 nutrition patterns in alzheimer’s disease 43 southeastern european medical journal, 2019; 3(1) introduction the number of people who are over 65 is growing and it is estimated that said age group will constitute up to 30% of the entire world population by the end of 2050. aging includes changes to all organs and body systems, as well as to the brain. in 2001, there were 24 million people in the world older than 60 and suffering from dementia. it is estimated that this number will double every twenty years and that by the end of 2040 80 million people will be suffering from it (1). the economic burden of care and treatment of neurodegenerative diseases also increases the need for seeking measures for prevention or reversing of age-related disorders (2). a neurodegenerative age-related disease like alzheimer’s (ad) appears to be the consequence of stress and of the same environmental factors that are responsible for the aging of other organs and body systems. the clear cause of the disease is still unknown, but some key changes, including membrane/synaptic degeneration and abnormal protein processing resulting in the formation of amyloid-β plaques and neurofibrillary tangles, have been detected (3). oxidative stress and inflammation are thought to play a significant role in the early stage of the disease, forming a vicious circle of damaging the sensitive brain cells. epidemiological studies point out that anti-inflammatory, antioxidant and neuroprotective agents found in food or plants may have a positive effect by strengthening the neuronal antioxidant defence. the mediterranean diet has also been linked with a lower incidence of neurodegenerative diseases such as ad (4). neurodegenerative diseases cannot be prevented using specific medication or treatment, so identifying a new natural compound with pharmacological properties is the subject of interest to a large number of scientists. chronic neurodegenerative diseases like ad and parkinson’s disease are related to obesity and diabetes, not just in the developing countries, but in the developed countries as well. dietary restrictions, including a diet low in saturated fats, cholesterol and carbohydrates and the consumption of berries, nuts and neuroprotective plant supplements, might have a positive effect on healthy brain functioning (5, 6). burgener et al. (2008) state that some people live to be 90 and have adequate cognitive functioning while some develop symptoms of ad in their late 50s. the difference might be due to interaction between genetics and environment (1). since we cannot fully affect genetics, this review will focus on actions promoting healthy brain aging and possible delay of the onset of the disease, including the consumption of certain foods, supplementation and introduction of dietary restrictions. methods literature research took place in january 2018. databases used in the search were pubmed, sciencedirect and springerlink. keywords neurodegenerative diseases, prevention, mediterranean diet, nutrition, supplementation were entered to identify papers dealing with neurodegenerative diseases and nutrition. the pubmed database yielded 2 articles. another 102 articles were found after using the option “similar results” with the first paper and 98 articles were found for the second paper. inclusion criteria all articles containing the terms alzheimer’s disease, neurodegenerative diseases, dementia, cognitive decline, mild, mediterranean diet, nutrition, vitamin b, c, e, d, antioxidants, supplementation, mufas, pufas, ω-3, ω-6, olive oil, prevention, cohort, prospective in the title or abstract were included in the review. if terms physical activity, cancer, cardiovascular diseases, respiratory problems, drugs appeared in the title or abstract of the article, they were excluded. other exclusion criteria were if a paper included animal research (dogs, rats), if it tested medicinal dietary supplements or if it could not be fully accessed. the criteria were ultimately met by 24 articles regarding studies conducted on 10 445 participants. seemedj 2019, vol 3, no. 1 nutrition patterns in alzheimer’s disease 44 southeastern european medical journal, 2019; 3(1) results the final results obtained after applying filters are shown in table 1. table 1. pubmed search methodlogy and results filters paper 1 paper 2 similar results 102 new articles 98 new articles from 2000 until 2018 97 98 on humans 92 96 english language 85 87 persons 65+ 16 21 review of abstracts and title of the articles based on including criteria 8 7 the same keywords were used to search the sciencedirect database and 430 articles were identified initially. this number was narrowed to 372 after setting the timeframe to include articles published from 2009 to 2018. the number of articles was further reduced to 69 by using the option “type of article – research” and to 11 by using the option “publication title”. they were published in the journals clinical nutrition, alzheimer’s & dementia and food research international. after reading the titles and abstracts, 4 articles were selected. the above-mentioned keywords were used for searching the springerlink database, where 264 articles were identified. after applying the filter article, the initial number was reduced to 177 and then to 23 after using the filter “biomedicine”. further restriction of results was accomplished using the options “neurosciences and english”, which led to 10 articles. titles and abstracts were reviewed, and 3 articles were selected. discussion neurodegenerative diseases and nutrition dry matter in the brain mostly consists of fat, 20% of which is made up of ω-3 and ω-6 polyunsaturated fatty acids, which significantly influence the structure and composition of the neuronal membrane. adequate nutrition is the source of those acids. aging brings changes in the taste and smell of food, the basal metabolic rate decreases, and due to possible swallowing impairment and impaired digestion, dietary intake is also reduced. unsaturated fatty acids and vitamins are thus in deficit. neuronal membrane lipids change and influence homeostasis necessary for membrane fluidity and function, as well as for prevention of loss of synaptic plasticity, apoptosis and neurodegeneration (3). since the cause of neurodegenerative diseases is still unknown, numerous researchers have made speculations regarding the connection between dietary habits, food intake and aging as the possible link for prevention or delay of the onset of neurodegenerative diseases, especially ad (7). particular attention was given to diets such as the mediterranean diet, food supplements and introduction of dietary restrictions. mediterranean diet notably, there are few published studies dealing with primary or secondary prevention of ad through nutritional interventions. most of them have focused on the effects of single nutrient supplementation (3). however, studies should focus on the entire dietary pattern and not just on a single nutrient in order to avoid neglecting important interactions between food components since people do not eat only single seemedj 2019, vol 3, no. 1 nutrition patterns in alzheimer’s disease 45 southeastern european medical journal, 2019; 3(1) nutrients (7). after analysing a number of dietary patterns, the mediterranean diet was selected. this type of diet is characterized by consumption of fruit and vegetables, legumes, fish and complex carbohydrates on a daily basis. olive oil is the main source of fat, and moderate drinking of red wine at meals is recommended (7). one of the first studies linking the mediterranean diet to a lower risk of developing ad was conducted in 2006 in new york on 2258 non-demented participants. after 4 years of follow-up, the results showed that higher adherence to this type of diet lowers the risk of developing ad, which indicates that that such diet may have a protective effect (8). anther cohort study, the washington heights/hamilton heights columbia aging project (whicap) conducted in 2012 on 1219 non-demented people, found that higher levels of ω-3 polyunsaturated fatty acids from fish, poultry, nuts and margarine were significantly associated with lower levels of plasma aβ42, which suggests that they play an important role in lowering the risk of ad and slowing cognitive decline (9). consumption of at least 100 grams of olive oil per day as one of the sources of polyphenols also slows cognitive decline (1). morris et al. (2015) studied the connection between the mind diet, a hybrid of the mediterranean diet, and the occurrence of ad. the mind diet contained 15 nutrients, 9 of which were linked to healthy brain aging (leafy greens, nuts, berries, legumes, whole grains, fish, poultry, olive oil, red wine) and 6 to unhealthy brain aging (red meat, margarine, cheese, pasta, sweets, fried food). the study was conducted on participants of the rush memory and aging project (map) in chicago from 2004 until 2014, which included 923 people aged 58 to 98. the results showed that even moderate adherence to the mind diet has a protective effect, significantly lowering the number of ad cases. this does not necessarily mean that the mind diet is specifically effective for ad, but that it has a protective effect on the brain and general functioning (10). fruit and vegetable consumption typical for the mediterranean diet showed a link with lower risk of ad and dementia in women, while consumption of fish and seafood significantly correlated with lower incidence of dementia in general among the people of southwestern france (11). the mediterranean diet, alone or in combination with other dietary patterns, represents the most effective approach to slowing the progression of ad with the fewest possible adverse effects (11). persons who adopt this type of diet are less likely to develop ad than their peers who do not (12). supplements and their role vitamins are important for normal functioning of the whole body, including the brain. therefore, the american food and drug administration agency mandated fortification of grains with folate from 1996 until 1997 to prevent vitamin deficiency (13). it is believed that a deficiency of folic acid and other b vitamins, along with vitamin c and e deficit, can contribute to the development of ad (14). however, results of vitamin supplementation are inconsistent. a longitudinal cohort study conducted on 965 people over 65 years in new york (northern manhattan) suggests that lower risk of ad development is associated with total folate intake from both food and dietary supplements, rather than intake from a single source, while intake of vitamins b6 or b12 is not linked to or significant for the risk of ad (13). similar results were obtained in a cross-sectional study conducted on 1219 cognitively healthy people older than 65, where no significant link was found between lower risk of ad and vitamin b9, b12, c, e and β-carotene supplementation (9). a longitudinal cross-sectional study conducted on 4470 participants (the cache county study) aimed to determine the link between antioxidant supplementation and the risk of ad development. previous studies reported that vitamins c and e act neuroprotectively and lower the risk of ad by scavenging free radicals and other reactive oxygen species (15). a longitudinal study in sweden involving 1810 people older than 75 (kungsholmen project), conducted from 1987 to 2000, identified that lower levels of vitamin b12 and folic acid, probably due to malnutrition or malabsorption in the elderly, double the risk of ad (16). vitamins b9 and b12, unlike vitamins c and e, regulate seemedj 2019, vol 3, no. 1 nutrition patterns in alzheimer’s disease 46 southeastern european medical journal, 2019; 3(1) homocysteine levels in the blood, which are associated with the risk of ad. a multicentre randomized double-blind controlled clinical trial conducted in america (adcs) from 2003 until 2006 on 340 participants reported that high dosage of vitamin b6, b12 and folic acid supplementation did not delay cognitive decline, nor did it improve the clinical status of persons with mild or moderate ad (17). the discrepancy of results may be due to the different mechanisms of action of vitamins in prevention of ad, the number of participants and their age, the type and duration of the study, preexisting vitamin deficiency and the type of prevention involved (primary, secondary or tertiary). berti et al. (2015) conducted a crosssectional study on 52 participants at risk of developing ad from 2013 to 2014 to clarify how nutrients are associated with healthy brain aging and prevention of ad. the objective was to identify how different patterns of nutrition affect the formation of aβ plaques (initial sign of pathological changes), glucose metabolism (indicator of neuronal activation) and grey matter volume of the brain (sign of atrophy). the results showed that higher intake of vitamins b12, d and zn reduces aβ plaque load in the brain, as well as increases neuronal activity and grey matter volume. higher activation of neurons was registered during higher intake of vitamin e, ω-3 and ω-6 polyunsaturated fatty acids, vitamins a, c and whole grains. intake of vitamins b 1, 2, 3, 6, 9 and minerals ca, fe, mg, p, k, se showed no significant effect on the formation of aβ plaques, neuronal activation and grey matter atrophy, while higher intake of saturated fatty acids, food high in cholesterol and sodium had a significant impact on lowering neuronal activation and grey matter volume (18). results obtained from a small number of carefully screened subjects in another large cohort study (three-city study) conducted in three french cities from 1999 and 2000 until 2012 on 9294 people, only 666 of whom were non-demented, confirmed that there is a strong link between lower intake of vitamin d, carotenes and polyunsaturated fatty acids, higher intake of saturated fatty acids and long-term risk of ad and dementia. future interventional research on primary prevention of ad and dementia should focus on the optimal combination of nutrients based on the results obtained so far (19). polyphenols this paper did not focus on the effects of polyphenols on animals, only on humans. vitamin p was first mentioned in 1936, and has been plentiful in the human diet since then. polyphenols are secondary plant products believed to have a positive effect on human health, particularly for the elderly (20). polyphenols from different fruit such as blueberries, blackberries, grapes and apples, from green and black tea, wine, coffee, cocoa and from spices such as turmeric and curry possess anti-inflammatory, antioxidant and neuroprotective properties (21). research on polyphenols in japan and singapore has indicated that they have the potential to be neuroprotective (12), although the underlying mechanisms have not been fully clarified. polyphenols interact with a wide range of neurotransmitters, suppressing neuroinflammation and degeneration through direct, indirect or complex action (22). since 1936, almost 10 000 articles have been published on the chemical nature and biological activities of polyphenols (20), suggesting that they have a neuroprotective effect. evidence from first-in-human research has identified that polyphenols have a positive effect, as part of a balanced diet. however, the effects of antioxidants have been disputed in the past few years. it has been suggested that their metabolites do not cross the blood-brain barrier, which brings their neuroprotective effect into question (20). caloric restrictions the average energy requirements for an adult person are 25 kcal/kg/day. they decline after adolescence because the human body stops growing and basal metabolic rate decreases. between the ages of 55 and 75 energy requirements decrease by 8% for every decade and by an additional 10% for every decade after 75 (23). caloric restriction as a way of primary seemedj 2019, vol 3, no. 1 nutrition patterns in alzheimer’s disease 47 southeastern european medical journal, 2019; 3(1) prevention of ad may be used only in those individuals with normal eating patterns. malnutrition and weight loss are usually associated with the onset of ad; caloric restriction would thus be counterproductive (23). discussion although no cure for neurodegenerative diseases exists, prevention and delaying the onset of the diseases is important. large cohort studies have confirmed the protective role of diets like the mediterranean diet, of a higher intake of polyunsaturated instead of saturated fatty acids, of a higher intake of fish and of vitamin supplementation (24). the role of decreased levels of a single nutrient and its supplementation in prevention of cognitive decline and dementia are eliciting controversy, probably because supplements act in a synergistic way and single nutrient supplementation will not lead to improvement (25). seven lifestyle risk factors have been identified in connection with ad. besides low education, physical inactivity, smoking, depression and hypertension, the role of nutrition is the most controversial and the role of sleeping disorders has not been sufficiently researched (26). a carefully controlled longterm interventional study on the effects of diet on prevention and delaying the onset of disease is required (24), with special consideration given to participants, type and duration of study, biomarkers for ad and intermediate outcomes (26). conclusion the most investigated nutrients in regard of ad and cognitive decline are ɷ-3 and ɷ-6 polyunsaturated fatty acids from fish, poultry, nuts and margarine, as well as, folic acid, vitamin c and e. consumption of polyphenol-rich foods like blueberries, blackberries, grapes, apples or beverages such as green and black tea, coffee and red wine may be beneficial. adherence to mediterranean diet is recommended because of the protective effect on the brain and general functioning. caloric restriction may be counterproductive in elderly. future intervention studies aiming to influence the modifiable lifestyle factors and to reduce the number of people at risk for ad, or at least slow down cognitive decline, are required. acknowledgments the authors would like to express sincere gratitude to professor sergej m. ostojić, md, phd, vice-dean for scientific research at the faculty of sport and physical education novi sad for his advice and instructions. they would also like to thank biljana rodić, md, phd, head of the neurology department and stroke unit of the cantonal hospital winterthur for the advice concerning neurodegenerative diseases. references 1. burgener sc, buettner l, buckwalter kc, beattie e, bossen al, fick dm, fitzsimmons s, kolanowski a, richeson ne, rose k, schreiner a, pringle specht jk, testad i, mckenzie s. evidence supporting nutritional interventions for persons in early stage alzheimer’s disease (ad). j nutr health aging 2008; 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(2013). mediterranean diet and dementia of the alzheimer type. curr aging sci 2013; 6:2, 150-162. 9. gu y, schupf n, cosentino sa, luchsinger ja, scarmeas n. nutrient intake and plasma β-amyloid. neurology 2012; 78:23, 1832-1840. 10. morris mc, tangney cc, wang y, sacks fm, bennett da, aggarwal nt. mind diet associated with reduced incidence of alzheimer's disease. alzheimers dement 2015; 11:9, 1007-1014. 11. shah r. the role of nutrition and diet in alzheimer disease: a systematic review. j am med dir assoc 2013; 14:6, 398402. 12. mallidou a, cartie m. nutritional habits and cognitive performance of older adults: anastasia mallidou and mario cartie look at the evidence concerning the effects of nutrition and hydration on cognition and alzheimer’s disease in older people. nursing management 2015; 22:3, 27-34. 13. luchsinger ja, tang mx, miller j, green r, mayeux r. relation of higher folate intake to lower risk of alzheimer disease in the elderly. arch neurol 2007; 64:1, 86-92. 14. bourre jm. effects of nutrients (in food) on the structure and function of the nervous system: update on dietary requirements for brain. part 1: micronutrients. j nut health aging 2006; 10:5, 377. 15. zand pp, anthony jc, khachaturian as, stone sv, gustafson d, tschanz jt, norton mc, welsh-bohmer ka, breitner jcs. reduced risk of alzheimer disease in users of antioxidant vitamin supplements. the cache county study. arch neurol 2004; 61:1, 82–88. 16. fratiglioni l, winblad b, von strauss e. prevention of alzheimer's disease and dementia. major findings from the kungsholmen project. physiol behav 2007; 92:1, 98-104. 17. aisen ps, schneider ls, sano m, diaz-arrastia r, van dyck ch, weiner mf, bottiglieri t, jin s, stokes kt, thomas rg, thal lj. high-dose b vitamin supplementation and cognitive decline in alzheimer disease: a randomized controlled trial. jama 2008; 300:15, 17741783. 18. berti v, murray j, davies m, spector n, tsui wh, li y, williams s, pirraglia e, vallabhajosula s, mchugh p, pupi a, de leon mj, mosconi l. nutrient patterns and brain biomarkers of alzheimer’s disease in cognitively normal individuals. j nutr health aging 2015; 19:4, 413-423. 19. amadieu c, lefèvre-arbogast s, delcourt c, dartigues jf, helmer c, féart c, samieri c. nutrient biomarker patterns and long-term risk of dementia in older adults. alzheimers dement 2017; 13:10, 1125-1132. 20. schaffer s, asseburg h, kuntz s, muller we, eckert gp. effects of polyphenols on brain ageing and alzheimer’s disease: focus on mitochondria. mol neurobiol 2012; 46:1, 161-178. 21. visioli f, burgos-ramos e. selected micronutrients in cognitive seemedj 2020, vol 4, no. 1 stigmatization of patients with viral infections and mass psychogenic illness 49 southeastern european medical journal, 2020; 4(1) decline prevention and therapy. mol neurobiol 2016; 53:6, 4083-4093. 22. almeida s, alves mg, sousa m, oliveira pf, silva bm. are polyphenols strong dietary agents against neurotoxicity and neurodegeneration? neurotox res 2016; 30:3, 345-366 23. ramesh bn, rao ts, prakasam a, sambamurti k, rao ks. (2010). neuronutrition and alzheimer's disease. j alzheimers dis 2010; 19:4, 1123-1139. 24. weih m, wiltfang j, kornhuber j. non-pharmacologic prevention of alzheimer’s disease: nutritional and lifestyle risk factors. j neural transm 2007; 114:9, 1187-1197. 25. rikkert o, marcel gm, verhey fr, sijben jw, bouwman fh, dautzenberg pl, lansinh m, sipers w, van asselt d, van hees a, stevens m, vellas b, scheltens p. differences in nutritional status between very mild alzheimer's disease patients and healthy controls. j alzheimers dis 2014; 41:1, 261-271. 26. carrillo mc, brashear hr, logovinsky v, ryan jm., feldman hh, siemers er, abushakra s, hartley dm, petersen rc, khachaturian as, sperling ra. can we prevent alzheimer's disease? secondary “prevention” trials in alzheimer's disease. alzheimers dement 2013; 9:2, 123-131. seemedj 2018, vol 2, no. 1 the use of hydrogen sulfide donor in cardiac control 36 southeastern european medical journal, 2018; 2(1) the use of hydrogen sulfide donor in cardiac control during low doses of ionizing radiation (experimental study)1 iryna kovalchuk1, mechyslav gzhegotskyi1, svitlana kovalchuk1, vasyl dukach1 1 department of physiology, danylo halytsky lviv national medical university, ukraine corresponding author: iryna kovalchuk, tarakanchikova@gmail.com received: dec 22, 2017; revised version accepted: nov 30, 2018; published: november 27. 2018 keywords: hydrogen sulfide h2s, sodium bisulfide, radiation effects, autonomic nervous system (ans), cardiovascular system (cvs), heart rate control abstract aim: to analyze the influence of the hydrogen sulfide donor nahs on the cardiac control in rats during low doses of ionizing radiation and characterize the predictors of imbalance in autonomic nervous system activities. methods: adult male rats were treated once with nahs 7.4 mg/kg, i.p. or vehicle (saline), without and with total body irradiation (tbi). the irradiation of animals in experimental groups was singlefractional, total, with total absorbed dose – 2 gy. heart rate variability (hrv) was recorded in rats after 30 minutes with nahs pre-treatment, 24 h of tbi exposure and nahs and tbi combination. results: hrv analysis revealed that low doses of radiation cause cardiac autonomic dysfunction. tbi rats exhibited signs of activation of sympathetic nervous system, while nahs-treated rats showed mobilization of all parts of regulatory systems with predominant activation of the parasympathetic nervous system (psns). overall, these results indicate that nahs is involved in cardiac control and decreases sensitivity to low doses of radiation. conclusion: the gaseous messenger h2s under conditions of radiation modulates adaptive processes, acting as a regulator for imbalance of autonomic nervous system and may serve as a novel cardioprotective agent for ans imbalance. (kovalchuk i, gzhegotskyi m, kovalchuk s, dukach v. the use of hydrogen sulfide donor in cardiac control during low doses of ionizing radiation (experimental study). seemedj 2018; 2(1); 36-43) seemedj 2018, vol 2, no. 1 the use of hydrogen sulfide donor in cardiac control 37 southeastern european medical journal, 2018; 2(1) introduction recent data on extracellular and intracellular control of biological activities of gas transmitter hydrogen sulfide (h2s) has shown that it plays multiple roles in several signaling pathways of physiological functions in almost all systems of the body [4, 12, 22]. according to the latest data, h2s is an important key regulator in the cardiovascular system [1, 17], neural signaling [27], gastrointestinal tract [13, 25], antiinflammatory activity [28], as well as metabolic disorders [29]. moreover, it has been established that the cardio-protective effect of hydrogen sulfide manifests in the reduction of myocardial damage during ischemia-reperfusion injury, oxidative stress, etc.[8, 20, 21]. h2s in physiological endogenous concentrations shows local [24, 25] and central effects (control of blood pressure and cardiac activity) [4, 12]. according to the other reports, h2s has been registered to have dose-dependent negative chronic and inotropic effects on the heart, which obviously depends on the initial state of the myocardium [23]. it is known that h2s can act as a free radical scavenger, reacting with radical oxygen species (ros) and radical nitrogen species (rns), including superoxide anion radical, peroxide, peroxynitrite and hypochlorite [5, 30, 14]. several studies have also been conducted to show mechanisms of regulation of the physiological functions involving higher levels of control with the participation of h2s. thus, the authors of the present study tried to analyze the impact of hydrogen sulfide on peripheral and central mechanisms involved in control of cardiac activity. one of the integrative tests for assessing the functional activity of different levels of regulatory systems was the monitoring of heart rate variability (hrv) [3, 7, 11, 16]. this non-invasive method enables not only high accuracy in assessing the activity of the autonomic nervous system (ans) in experimental conditions, but also reflects the physiological and biochemical regulatory mechanisms, the capacity of aerobic metabolism, as well as the physiological reserve capabilities of the physiological system [15, 18, 19]. due to its high reducing properties, h2s participates in the regulation of changes in oxidative and nitrosative stress [11, 14, 17]. one of the factors that causes oxidative stress is the effect of ionizing radiation. natural and manmade sources of ionizing radiation contribute to human exposure and, accordingly, increase potential risk to human health. much radiation is unavoidable, e.g., natural radiation, and represents a potential health risk [6]. as known, the human body responds to various physical, chemical and biological stimuli by developing a potent inflammatory response that triggers defense mechanisms crucial for maintaining tissue integrity and restoring tissue homeostasis and functions. ionizing radiation-induced systemic effects usually arise from local exposure of an organ or part of the body [10]. the target for low doses of radiation are the membranes of subcellular and cellular structures, in which free radical processes are induced. for experimental rats, who are highly radioresistant animals (ld50 7 – 7.5 gy), the dose of 2 gy is considered low [2]. relatively little work has been done on this topic associated with the effects of h2s and its main mechanisms involved in the systemic actions of ionizing radiation on the level of the whole body or on cellular level. recently, it has been shown that low-dose radiation has a harmful systemic effect on the human body [6, 10, 26], but the influence of nahs pretreatment during ldr injury is still not clear. therefore, the aim of this study was to analyze the effects of exogenous administration of hydrogen sulfide donor nahs on cardiac control in rats under ldr and implement a comprehensive analysis of these changes in the parameters of hrv. materials and methods twenty-five male rats with an average weight 180-200 g that were used in our study were kept in conditions that were in accordance with university policies, and all experiments were approved by the ethics committee of lviv national medical university (20.01.2015; №1). the animals were selected into experimental groups seemedj 2018, vol 2, no. 1 the use of hydrogen sulfide donor in cardiac control 38 southeastern european medical journal, 2018; 2(1) (n=5): 1) control group – rats with single administration of 0.9% solution of nacl at a dose 0.5 ml, i.p.; 2) group with single administration of nahs at a dose of 7.4 mg/kg (sigma aldrich, usa), i.p. and registration of hrv values in 30 minutes; 3) group with administration of nahs at the same dose and determination of hrv in 1 day; 4) group that underwent irradiation in the total absorbed dose of 2 gy; 5) group with introduction of nahs + irradiation (2 gy). animals of the experimental group were irradiated with a single-fractional teletherapy machine device "teragam" (source 60со) at a dose rate of 0.0393 r/s and a "source-surface" of 0.8 m. the total absorbed dose was 2 gy. during irradiation, the animals were placed in individual cagesfixators. we recorded a peripheral pulse non-invasively in non-anaesthetized animals for the purpose of hrv analysis (hzhehotskyi mr, patent ua, 2008) [9]. to record the parameters, 5 minutes after the rats stabilized, they were placed in a plexiglas chamber and a photoplethysmographic transducer was attached to the base of each animal's tail [9]. measuring of hrv parameters was carried out before the administration of nahs, after 30 minutes and one day after the procedure, as well as one day after irradiation. changes in hrv parameters were compared to their baseline values (before manipulations), which served as a control. the duration of cardiointervals (averval interv) was determined using software and a special fast-acting recording device [9]. during recording, dynamic series of cardiointervals were presented in the form of a cardiointervalograms. there was simultaneous analysis of dynamic series of cardiointervals and interpretation of hrv analysis data. the activity of the system-regulatory mechanisms of experimental animals was evaluated on the basis of spectral statistical methods, as well as variation pulsometry. among the statistical parameters of the dynamic series of cardiointervals, the following indexes were determined: sdnn – the mean square deviation of the mean cardiointerval duration, cv – the coefficient of variation of the full array of cardiointervals, which characterizes the total effect of autonomous blood flow regulation; rmssd – the square root of the sum of the differences in the series of cardiointervals, which was an indicator of activity of the parasympathetic link of autonomous regulation [3, 18]. among the parameters of the variational pulsometry, the following ones were determined: the difference between the maximum and minimum values of cardiointervals (mxdmn) – the maximum amplitude of regulatory influences; mode (mo) – the most likely level of functioning of the sinus node of the heart; mode amplitude (amo) was a conditional indicator of the activity of the sympathetic link of regulation [3, 18]. with the help of spectral analysis, it was possible to characterize such parameters as the total power of the hrv spectrum (tp, 0.015 – 3.0 hz) – the total absolute level of activity of regulatory systems [3, 18]. for the correct reproduction of the results of studies conducted in laboratory animals, the analysis of hrv was carried out using spectral components in the following frequency bands: low frequency – lf (0.0150.25) hz, which was associated with the level of activity of ergotropic slow influences, mid frequency – mf – (0.25-0.75) hz, caused by sympathetic modulating effect on the cardiovascular system, high frequency – hf – (0.75-3.0) hz, which was characteristic of the activity level of the parasympathetic link of regulation [20, 21]. the digital results of all measurements were subjected to statistical variation analysis. the results were processed using microsoft excel spreadsheets and statistical software statistica for windows. results as a result of the conducted analyses, it was found that the total power of the spectrum has increased to 78% in comparison to the basal level (control group) due to the increase of spectral power in all frequency bands, significantly 30 minutes after the administration of the h2s donor (figure 1). analysis of the internal structure of the spectrum has shown seemedj 2018, vol 2, no. 1 the use of hydrogen sulfide donor in cardiac control 39 southeastern european medical journal, 2018; 2(1) increased proportion of hf (24% (p<0.05)) and tendency of decreasing proportion of mf and lf. correspondingly, the sympathovagal index (mf/hf) decreased to 26% (<0.05) vs the control group (figure 1). figure 1. changes in hrv spectrum indexes under the radiation exposure and previous administration of nahs vs control group, which is marked as 100 % (blue line). note: *– p<0.05 significance in relation to the control group; • – p<0.05 significance in relation to the irradiation group. also, the increased spectral power in the range of high frequency oscillations was detected, which correlates with rise of cv to 75% (p<0.05), sdnn to 32%, rmssd to 41% (p<0.05), and mxdmn to 53% (p<0.05)). in general, this testifies to the activation of the autonomous circuit of regulation of cardiac activity (table 1). decreased amо (18% (p<0.05)), as well as mf/hf in comparison to control group indicates inhibition of sympathetic nervous system (sns) activity. at the same time, a positive chronotropic effect was recorded, which may result in an increase in cardiac output (table 1). seemedj 2018, vol 2, no. 1 the use of hydrogen sulfide donor in cardiac control 40 southeastern european medical journal, 2018; 2(1) table 1. changes of hrv in rats parameters control nahs 30 min nahs 1st day irradiation 1st day nahs + irradiation 1st day hr (heart rate), bpm 431.4±27.3 539.3±39.7 444.7±29.7 433.1±31.2 406.1±24.7 sdnn (mean square deviation of the mean cardiointerval duration), s 0.030±0.003 0.039±0.003 0.037±0.002 0.024±0.002 0.029±0.002 cv (coefficient of variation), % 22.1±19 38.6±2.8 27.9±2.3  17.7±1.3 19.0±1.5 rmssd (square root of the sum of the differences in series of cardiointervals), s 0.031±0,003 0.043±0.004 0.038±0.003 0.025±0.002 0.026±0.002 mo (mode), s 0.151±0.012 0.151±0.014 0.151±0.013 0.147±0.012 0.151±0.013 amo (mode amplitude), % 37.1±3.1 30.4±2.7 25.4±2.1  36.3±3.2 37.2±3.4 mxdmn (maximum and minimum values of cardiointervals), s 0.128±0.011 0.196±0.017 0.148±0.013 0.138±0.012 0.122±0.014 note:  – р0.05 significance in relation to the control group. the results of hrv 24 h after administration of nahs have shown noted maintenance of a high level of tp which exceeded the control value on 37% (p<0.05), however, it decreased on about 23% (p<0.05) after the influence of hydrogen sulfide on the 30th minute (figure 1). as in the previous term, the spectral power increased the most in the range of highfrequency oscillations, both in comparison with the initial level and the level measured 30 minutes after the influence of nahs. the increase of statistical parameters of hrv as cv, sdnn, rmssd, as well as mxdmn (on average 25% (p<0.05) vs control data) was also noted, which indicates an increase in the tonus of the psns, but it was of a lesser degree of severity than data recorded at the 30th minute (table 1). one day after tbi at a dose of 2 gy, the total power of spectrum (tp) decreased twofold compared to the control group (figure 1). the decrease in power in all spectrum ranges was recorded: lf and mf to 54% and 52% (p<0.05) respectively, hf – to 40% (p<0.05). this data suggests a significant inhibition of the activity of all parts of cardiac regulatory systems. in that seemedj 2018, vol 2, no. 1 the use of hydrogen sulfide donor in cardiac control 41 southeastern european medical journal, 2018; 2(1) case, in the internal structure of the spectrum, the proportion of lf decreased to 16%, and mf practically did not change. the reduction of hrv time parameters (sdnn, cv, rmssd) was marked (20% on average (p<0.05)), which indicates inhibition of psns activity (table 1). in terms of the influence of irradiation accompanied by administration of nahs, it was found that the values of time indexes have increased significantly relative to irradiation and almost reached the baseline (table 1). thus, the total power of the spectrum 1 day after irradiation accompanied by administration of a donor hydrogen sulfide was reduced to 27% relative to the results of control, but it was significantly higher than at single irradiation (figure 1). at the same time, the spectral power in the range of lf and mf, which was 86% vs control, was significantly increased compared to the rats with tbi effect. discussion the obtained data are consistent with the results of other researchers, which have established the cardioprotective effect of h2s, manifested by an increase in the strength of cardiac contractions, as well as vasodilator effect in the period after the administration of a donor hydrogen sulfide [12, 13]. the increase in tp, as well as the spectral power in the hf and mf, rmssd and other hrv statistical indexes, the decrease in mf/hf can be regarded as an increase in functional and metabolic reserves under the influence of h2s. consequently, the revealed complex array of changes in the parameters of hrv indicates the mobilization of all regulatory systems with the predominant activation of the parasympathetic nervous system (psns) after the administration of nahs [8, 11]. an elevated level of balanced autonomous components (hf + mf) and decrease of mf/hf index was a prominent marker of influence of peripheral regulatory system on central control. the obtained results of the study indicated a protective effect of nahs under influence of ldr, which indirectly indicates improvement of processes of adaptation to the action of radiation under the influence of hydrogen sulfide biosynthesis modification [19], which consisted of activation of various links of regulatory systems with prevailing mobilization of the parasympathetic nervous system and moderate activation of peripheral control system regulated by h2s. on the 1st day after administration of nahs, there was a prolonged effect in comparison to that found in the initial (30 min.) period of exposure to the hydrogen sulfide donor, nahs, which ensures the maintenance of increased activity of regulatory processes. thus, the reduction of the total effect of autonomic regulation by sdnn, rmssd and cv correlated with the reduced total level of activity of regulatory processes by the spectral index of tp and indicated the activation of central regulatory systems. the previous administration of nahs before irradiation led to a less pronounced reduction in tp than with irradiation alone, indicating the maintaining of a much higher level of regulatory processes in these conditions than with irradiation alone. the effect of donor hydrogen sulfide nahs against low-dose radiation injury, which was reflected in the modulation of adaptive processes, acting as a regulator of imbalance of autonomic nervous system, may serve as a novel cardioprotective agent increasing the capacity of restorative processes. conclusion in conclusion, the data of the present study support that exogenous stimulation of peripheral h2s-dependent signaling processes by administration of nahs significantly affected the systemic regulatory mechanisms of cardiac activity on different levels. after 30 minutes and 1 day following the administration of the donor hydrogen sulfide, activation of all the links of regulatory systems with the predominant mobilization of psns was marked. one day after irradiation at a dose of 2 gy, the tp decrease was twofold, with decreasing power in all ranges, indicating significant inhibition of the activity of all regulatory systems. finally, irradiation accompanied by previous administration of nahs leads to a less pronounced reduction in tp seemedj 2018, vol 2, no. 1 the use of hydrogen sulfide donor in cardiac control 42 southeastern european medical journal, 2018; 2(1) than with irradiation alone, indicating the maintaining of a higher level of regulatory processes. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare references: 1. apaijai n, pintana h, chattipakorn sc et al. effects of vildagliptin versus sitagliptin, on cardiac function, heart rate variability and mitochondrial function in obese insulin-resistant rats. br j pharmacol 2013;169:1048–1057. 2. asrar m. havas. the biosensitivity of certain organs in rats exposed to low doses of γ -radiation. 2013;6:56-62. 3. baevsky rm, ivanov gg, chirejkin lv et al. the analysis of heart rate variability with use various electrocardiographical systems. the bulletin of arrhythmology 2001;24:65-87. 4. berezovsky vj, plotnikova lm. hydrogen sulfide and its role in the vascular tone regulation. med hydrol and rehabilit 2012;10:4– 10. 5. cipak gasparovic a, zarkovic n, zarkovich k, semen k, kaminskyy d, yelisyeyeva o, bottari sp. biomarkers of oxidative and nitro-oxidative stress: conventional and novel approaches. br j pharmacol 2017;174(12):1771–1783. doi: 10.1111/bph.13673 6. tang fr, loke wk. molecular mechanisms of low dose ionizing radiationinduced hormesis, adaptive responses, radioresistance, bystander effects, and genomic instability int j radiat biol 2015;91(1):13-27. doi: 10.3109/09553002.2014.937510. 7. hillebrand s1, gast kb, de mutsert r, swenne ca, jukema jw, middeldorp s, rosendaal fr, dekkers om. heart rate variability and first cardiovascular event in populations without known cardiovascular disease: metaanalysis and dose-response meta-regression. europace 2013;15(5):742-9. doi: 10.1093/europace/eus341. 8. hoshovs’ka jv, shymans’ka tv, semenychina om, sagach vf. cardioprotective effects of hydrogen sulfide donor. fiziol zh 2012;58(6):3-15. 9. hzhehotskyi mr, storchun yv, panina lv, kovalchuk sm et al. method for evaluation of experimental animal functional state based on heart rate variability test. patent 29596 ua, 2008. http://uapatents.com/5-29596-sposib-ocinkifunkcionalnogo-stanu-eksperimentalnikhtvarin-na-osnovi-analizu-variabelnostisercevogo-ritmu.html 10. mavragani iv, laskaratou da, frey b, candéias sm, gaipl us, lumniczky k, georgakilas ag. key mechanisms involved in ionizing radiation-induced systemic effects. a current review. toxicology research 2016;5:1233. 11. jeppesen j, fuglsang-frederiksen a, brugada r, pedersen b, rubboli g, johansen p, beniczky s. heart rate variability analysis indicates preictal parasympathetic overdrive preceding seizure‐induced cardiac dysrhythmias leading to sudden unexpected death in a patient with epilepsy. epilepsia 2014;55(7):e67-71. doi: 10.1111/epi.12614. 12. kimura h. hydrogen polysulfide signaling along with hydrogen sulfide (h2s) and nitric oxide (no). j neural transm 2016;123(11):1235-45. 13. magierowski m, magierowska k hubalewska-mazgaj m, adamski j, bakalarz d, sliwowski z, pajdo r, kwiecien s, brzozowski t. interaction between endogenous carbon monoxide and hydrogen sulfide in the mechanism of gastroprotection against acute aspirin-induced gastric damage. pharmacol. res 2016;114:235-250. doi: 10.1016/j.phrs.2016.11.001 14. mancardi d, penna c, merlino a, del soldato p, wink da, pagliaro p physiological and pharmacological features of the novel gasotransmitter: hydrogen sulfide. biochimica et seemedj 2018, vol 2, no. 1 the use of hydrogen sulfide donor in cardiac control 43 southeastern european medical journal, 2018; 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6(2) original article vitamin d deficiency among medical students in osijek, croatia 1 stipe vidović 1*, željko debeljak 2, tatjana bačun 3,4, sven viland 1, milorad zjalić 1, marija heffer 1 1 department of medical biology and genetics, faculty of medicine, j. j. strossmayer university of osijek, osijek, croatia 2 clinical medical center osijek, institute of clinical laboratory diagnostics, croatia 3 clinical medical center osijek, department of endocrinology, osijek, croatia 4 j. j. strossmayer university of osijek, faculty of medicine osijek, department of internal medicine and history of medicine, osijek, croatia *corresponding author: stipe vidović, stipevidovic1@gmail.com received: oct 18, 2022; revised version accepted: nov 6, 2022; published: nov 28, 2022 keywords: vitamin d deficiency; 25-hydroxyvitamin d concentration; medical students; hypovitaminosis d abstract aim: this study aimed to evaluate serum levels of 25-oh d3 (calcidiol) among students of the faculty of medicine osijek, croatia, thereby determining to what extent vitamin d deficiency is present. methods: the present cross-sectional analysis was based on data collected from 60 participants. blood sampling was done in march 2021. concentrations of 25-oh d3 were measured using lc/msms procedure on shimadzu lcms 8050 and recipe kit for serum levels of 25-oh-d3. results: the study was conducted on a sample of 60 respondents aged 19 to 28, of whom 16 were men and 44 were women. all subjects had a 25-oh d3 deficiency (<20 ng/ml), while 11 had an extreme deficiency. mean 25-oh d3 concentration was 11.36 ng/ml, ranging from 7.70 ng/ml to 16.70 ng/ml. there was no statistical significance of 25-oh d3 concentration levels between the sexes (student’s t-test, p>0.05). conclusion: vitamin d deficiency was detected in all subjects. in addition to the results of several other studies conducted worldwide that evaluated vitamin d status among medical students, this study further highlights the problem affecting this student subgroup. (vidović s*, debeljak ž, bačun t, viland s, zjalić m, heffer m. vitamin d deficiency among medical students in osijek, croatia. seemedj 2022; 6(2); 67-74) seemedj 2022, vol 6, no 2 vitamin d deficiency among medical students 68 southeastern european medical journal, 2022; 6(2) introduction over the past decade, there has been a growing number of studies focusing on vitamin d and its roles in human health and disease. initially, it was thought to be only essential for rickettsial disease prophylaxis; however, contemporary research suggests the importance of vitamin d in maintaining individuals' well-being and overall health (1,2). the active form of vitamin d, 1,25dihydroxycholecalciferol (1,25-oh d3), has a pivotal role in maintaining mineral homeostasis and enhancing absorption of calcium and phosphate into the extracellular fluid by acting on the intestines, kidneys, and bones. therefore, it regulates bone growth and reorganization by interfering with the activity of osteoclasts and osteoblasts (3). vitamin d receptors are expressed in almost all cell types, which may explain the multiple actions of vitamin d on different tissues (4). although the proportion of the role of vitamin d and its effects on non-skeletal health is still debatable, it is associated with regulation of the innate and adaptive immune systems, reduction of inflammation, cell proliferation and differentiation, insulin and glucose secretion regulation, preventive effects on cardiovascular and neurodegenerative diseases, and even antiaging effects (5–9). for most people, the primary source of vitamin d is skin exposure to ultraviolet radiation, specifically uvb rays, where 7dehydrocholesterol is converted to pre-vitamin d3 in the skin during exposure to uvb rays (10). however, it can also be obtained through diet and dietary supplements (11). vitamin d testing has increased significantly in recent years, establishing the presence of vitamin d deficiency on a pandemic scale worldwide (12). it is estimated that more than a billion people worldwide have low vitamin d levels, with extensive observational data indicating that 40% of the european population and 24% of the us population have 25-oh d3 below 20 ng/ml (13). vitamin d deficiency can be diagnosed by measuring serum levels of 25-hydroxyvitamin d3 (14,15). although there is no consensus on optimal levels of 25 oh-d3, most guidelines suggest that concentrations below 20 ng/ml indicate vitamin d deficiency (16). (table 1). table 1. recommended serum levels of 25-oh d3 for verification of deficiency and adequate levels of vitamin d; according to croatian guidelines for the prevention, detection and therapy of vitamin d deficiency in adults (11). 25-oh d3 (ng/ml) interpretation <10 ng/ml extreme deficiency of vitamin d <20 ng/ml deficiency of vitamin d <30 ng/ml insufficiency of vitamin d ≥30 ng/ml adequate level of vitamin d >100 ng/ml excess >150 ng/ml intoxication in 2016, guidelines for preventing, detecting and treating vitamin d deficiency in adults were issued in croatia, according to which recommended levels of 25-oh d3 are between 30 and 100 ng/ml (17). numerous conditions are associated with vitamin d deficiency, such as chronic diseases, obesity, and malabsorption syndromes. it also occurs during pregnancy. in addition, skin pigmentation, clothing style, diet, sunscreen seemedj 2022, vol 6, no 2 vitamin d deficiency among medical students 69 southeastern european medical journal, 2022; 6(2) use, the season of the year, and latitude can also affect vitamin d synthesis and its serum levels, potentially leading to its deficiency, which increases the risk of developing skeletal and non-skeletal diseases (11,18). this study was aimed at evaluating serum levels of 25-hydroxyvitamin d3 among medical students of the faculty of medicine osijek in march 2021, thereby also determining to what extent vitamin d deficiency might be present among future healthcare professionals. they are potentially at a greater risk of developing hypovitaminosis d due to their work-related lifestyle, which could be even aggravated by the currently present covid-19 pandemic. material and methods the present cross-sectional analysis was based on data from 60 participants aged 19 to 28. blood sampling was done in march 2021 at the faculty of medicine in osijek and then analyzed at the clinical hospital center osijek, croatia. informed consent was obtained from all study participants. the research was approved by the ethics committee of the faculty of medicine osijek, university of osijek (class: 602-04/2208/02. no: 2158-61-46-22-149). subjects' blood was drawn into round bottom, polystyrene test tubes, 14 ml, 17 x 100 mm (becton dickinson, croatia) to separate serum from cells. the blood was centrifuged at 3000 rpm for 10 minutes. after centrifugation, the serum was separated into other tubes using disposable tubes, which were stored in a refrigerator at -20 ° c until analysis. according to the manufacturer's instructions (shimadzu corp.), the sample was analyzed on an lcms8050 analyzer. the lcms-8050 analytical system (shimadzu, japan) itself consists of nexera x2 hplc (high-performance liquid chromatography) system connected to a mass spectrometer (ms). the lc-ms / ms method is characterized by high sensitivity, specificity, and the possibility of simultaneous analysis of multiple analytes. it is considered the gold standard for determining vitamin d concentration in serum. statistical analysis numerical data was described by arithmetic mean and standard deviation. normality of distribution of numerical variables was determined by using the d'agostino-pearson test. differences in normally distributed numerical variables between two independent groups were assessed by using the student's ttest. all p values were two-sided. the significance level was set to alpha = 0.05. the medcalc statistical software version 20.106 was used for statistical analysis (https://www.medcalc.org/). table 2. sample age, gender and 25-oh d3 concentrations (ng/ml) (n=number, sd=standard deviation). total number of participants 60 male 16 female 44 male: female (ratio) 1:2.75 age (males), mean(sd) 21.9(1.2) age (females), mean(sd) 21.7(2.2) age (years), mean(sd) 21.9(1.2) 25-oh d3 levels (ng/ml) n 60 mean ±sd 11.37(1.59) minimum 7.67 maximum 16.68 25-oh d3 levels; males (ng/ml) n 16 mean (sd) 11.13(1.69) minimum 7.67 maximum 13.78 25-oh d3 levels; females (ng/ml) n 44 mean (sd) 11.46(1.53) minimum 8.29 maximum 16.68 https://www.medcalc.org/ seemedj 2022, vol 6, no 2 vitamin d deficiency among medical students 70 southeastern european medical journal, 2022; 6(2) results the study was conducted on a sample of 60 respondents aged 19 to 28, of whom 44 (73.33%) were women and 16 (26.67%) were men. all subjects had a 25-oh d3 deficiency (<20 ng/ml), with 11 (18.33%) of them exhibiting an extreme deficiency according to recommended serum levels of 25-oh d3 (17). the mean 25-oh d3 concentration was 11.36 ng/ml, ranging from 7.70 ng/ml to 16.70 ng/ml. the mean 25-oh d3 concentration in women was 11.45 ng /ml, while in men it was 11.13 ng/ml (table 2). we examined differences in 25-oh d3 concentration levels with respect to gender. the results showed no statistical significance of 25oh d3 concentration levels between the sexes (student’s t-test, p>0.05). pearson's correlation coefficient suggests that the relationship between age and 25 oh-d3 concentration is not significant (r=0.06; p>0.05).. figure 1. this scatter graph shows the 25-oh d3 status in the examined sample of 60 participants; expressed in ng/ml. the dashed black line indicates the threshold of vitamin d deficiency, while the solid black line indicates the threshold of extreme vitamin d deficiency. all subjects were vitamin d deficient (<20 ng/ml), where 11 (18.33%) of them had an extreme deficiency (<10 ng/ml). discussion according to our knowledge, this is the second study in croatia examining vitamin d levels among medical students. a study conducted in rijeka, croatia established a deficiency in half of the respondents, while we found hypovitaminosis among all of the participants (19). the results raise the question about the underlying cause and about the necessity of implementing prophylactic measures for this population in particular. the incidence of hypovitaminosis d increased in certain subpopulations due to various risk factors, such as lack of sun exposure, use of sunscreen, long hours indoors, latitude, diet, age, and various chronic and acute diseases (11). a 2017 meta-analysis emphasized the presence of an occupational risk among healthcare professionals, specifically medical students and residents (20). seemedj 2022, vol 6, no 2 vitamin d deficiency among medical students 71 southeastern european medical journal, 2022; 6(2) several studies worldwide involved measuring vitamin d levels among medical students and found a significant prevalence of vitamin d deficiency. in a study conducted in spain in 2011, 62% of students had lower than recommended levels of vitamin d (21). kardelen et al. determined vitamin d deficiency in 92% of medical students from turkey, while nadeem et al. found that only 13.6 % of healthy young medical students in pakistan had vitamin d levels within the normal range (22, 23). a study conducted in boston, usa in 1999 found deficiency among 34% of medical students (24). contrary to those findings, leary et al. found a low presence of hypovitaminosis among medical students in the us; only 5% of the subjects were deficient in florida and 13% in pennsylvania (25). in 2018, a study was also conducted in osijek, croatia, in the same period of the year as the current study. it also involved the measuring of vitamin d levels among young people aged 19 to 28. as in our study, a high incidence of hypovitaminosis was found, with 72.2% of the subjects having lower levels than 20 ng/ml. judging by those findings, a high incidence of hypovitaminosis was present. however, a deficiency was significantly less present than in our study, where all of the subjects were medical students (26). circumstances that may have affected vitamin d levels sampling was carried out in march, when vitamin d levels are lower due to insufficient sun exposure and seasonal variation in the intensity of solar uv light (27). research indicates that vitamin d concentrations for all age groups in central europe begin to decrease in early september and increase in late may (28). in this context, it has been found that medical students in croatia do not get enough sunlight during the day (29). a diet poor in vitamin d is another risk factor that may contribute to its low levels. a study from the uk found that students do not get enough vitamin d through their diet, where only 14% of respondents met recommended intake levels (30). a similar trend was noticed among students in china (31). in addition, a 2019 study found that medical students in rijeka, croatia, get only a fifth of the recommended vitamin d intake through diet (29). the period of the covid-19 pandemic is another circumstance that may affect vitamin d levels in this sample. the croatian government, like many others, has implemented a series of measures to reduce the number of people affected by covid-19, including social distancing and recommendations to stay at home, which could potentially limit sun exposure and in turn contribute to lowering vitamin d levels. yamaguchi et al. found a significant increase in hypovitaminosis d among healthcare workers during the covid-19 pandemic, where about 90% of the sample was deficient in vitamin d (32). this is essential, assuming that participants had a similar lifestyle in that period as our study sample, considering the duration of indoor activities in medical care and daily life routines. also, all study respondents live in the area around 45° latitude. latitude is considered a statistically significant risk factor for vitamin d deficiency in the sense that it is inversely proportional to the level of circulating 25-oh d3 (10). leary et al. found that medical students at higher latitudes had lower vitamin d levels. in that study, subjects (also medical students) were located at a similar latitude (42°) as in our sample; but compared to our results, the prevalence of hypovitaminosis was significantly lower — 13 % of participants were vitamin d deficient (25). vitamin d supplementation a 2018 study found that 30.5% of croatian students practice some form of supplementation, among which medical students used almost twice as much as others (33). jovanović et al., established that very few of their subjects (4.7%) used vitamin d supplementation (19). cholecalciferol, ergocalciferol, calcidiol, and calcitriol are used; they are acceptable and straightforward replacements for lack of sun exposure and insufficient intake through diet. considering seemedj 2022, vol 6, no 2 vitamin d deficiency among medical students 72 southeastern european medical journal, 2022; 6(2) different pharmacokinetic characteristics, metabolism, and points of regulation, cholecalciferol, known as vitamin d3, is most often recommended and used to prevent and treat deficiency (34). although much has been said about the importance of preventive use and optimal vitamin d supplementation in health and disease, there is no uniform consensus on the recommended daily intake. the institute of medicine and the endocrine practice guideline committee defined specific guidelines for the general population, where recommended intake was 600 iu/day for people aged 1 to 70, with the upper tolerable intake limit of 4000 iu/day (16). in 2016, croatian guidelines for the prevention, detection and therapy of vitamin d deficiency in adults defined "vitamin d therapy for verified deficiency, depending on age and comorbidities"(17). according to the guidelines, vitamin d deficient people over 18 years of age without other comorbidities should take vitamin d supplementation of 6000 iu/day for eight weeks, then maintain with 1500-2000 iu/day. considering that all subjects were deficient, following the previously mentioned recommendations could be one of the effective ways of optimizing vitamin d levels in the serum. conclusion in addition to the results of several other studies conducted worldwide that evaluated vitamin d status among medical students, this study further highlights the problem affecting this student subgroup. also, it would be helpful to examine vitamin d levels among medical doctors who, through education, should have gained insight into the concept of a healthy lifestyle and proper prevention methods of various conditions, including hypovitaminosis. study limitations the limitation of this study is that it was conducted on a small sample. also, testing was done only once in the winter period. moreover, there is a lack of assessment of participants' lifestyles correlated with vitamin d status, medical history of vitamin d-related diseases, and vitamin d supplementation. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. chanchlani r, nemer p, sinha r, nemer l, krishnappa v, sochett e, et al. an overview of rickets in children. kidney int rep. 2020; 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117(7):433–9. 26. šarić j. koncentacija vitamina d u muškaraca i žena u dobi od 18 do 28 godina u zimskom radoblju [internet] [info:eurepo/semantics/masterthesis]. josip juraj strossmayer university of osijek. faculty of medicine osijek; 2019 [cited 2022 may 17]. available from: https://urn.nsk.hr/urn:nbn:hr:152:855195 27. heidari b, haji mirghassemi mb. seasonal variations in serum vitamin d according to age and sex. casp j intern med. 2012; 3(4):535–40. 28. pludowski p, grant wb, bhattoa hp, bayer m, povoroznyuk v, rudenka e, heorhi ramanau h, varbiro s, rudenka a, karczmarewicz e, lorenc r, czechkowalska j, konstantynowicz j. vitamin d status in central europe. int j endocrinol. 2014; 2014:589587. 29. kenđel jovanović g, krešić g, pavičić žeželj s. dietary vitamin d intake among university students and their habits concerning daily sunlight exposure – a cross-sectional study. med flum. 2021 ;57(4):396–406. 30. dong h, asmolovaite v, marseal n, mearbon m. vitamin d status and dietary intake in young university students in the uk. nutr food sci. 2022; 52(4):616–26. 31. zhou m, zhuang w, yuan y, li z, cai y. investigation on vitamin d knowledge, attitude and practice of university students in nanjing, china. public health nutr. 2016; 19(1):78–82. 32. funaki t, sanpei m, morisaki n, mizoue t, yamaguchi k. serious vitamin d deficiency in healthcare workers during the covid-19 pandemic. bmj nutr prev health. 2022; 5(1):134–6. 33. žeželj s, tomljanović a, jovanović g, krešić g, peloza o, dragaš-zubalj n, pavlinić prokurica i. prevalence, knowledge and attitudes concerning dietary supplements among a student population in croatia. int j environ res public health. 2018; 15(6):1058. 34. vieth r. vitamin d supplementation: cholecalciferol, calcifediol, and calcitriol. eur j clin nutr. 2020; 74(11):1493–7.. author contribution. acquisition of data: sv, žd, tb,mh administrative, technical or logistic support: sv, žd, tb, s viland, mz, mh analysis and interpretation of data: sv, žd, tb, mz, mh conception and design: sv, tb, mz, mh critical revision of the article for important intellectual content: sv, žd, tb, mh drafting of the article: sv, žd, mh final approval of the article: sv, žd, tb, mh guarantor of the study: tb, mh obtaining funding: tb, mh provision of study materials or patients: žd, tb, mh statistical expertise: sv, mh seemedj 2017, vol 1, no. 2 isotope studies of karst springs included in the water supply system… 46 southeastern european medical journal, 2017; 1(2) isotope studies of karst springs included in the water supply system of the city of rijeka (croatia) 1 diana mance1,2, danijela lenac3, josip rubinić4 1 physics department, university of rijeka, rijeka, croatia 2 stable isotope laboratory, physics department, faculty of medicine, university of rijeka, rijeka, croatia 3 water supply company, rijeka, croatia 4 faculty of civil engineering, university of rijeka, croatia corresponding author: diana mance – diana.mance@uniri.hr received: september 21, 2017; revised version accepted: november 24, 2017; published: november 24. 2017 keywords: drinking water, isotopes, radioisotopes conclusion. study results showed that agnor count and structure cannot help in determining more clearly the border between parathyroid adenoma and hyperplasia in cytologic smears. (fustar preradovic lj, danic d, kardum-skelin i, sarcevic b, danic hadzibegovic a. agnor counts in differential diagnosis of parathyroid adenoma and hyperplasia in preoperative cytologic smears. seemedj 2017; 1(2); 19-26) abstract aim: to provide information about the results of isotopic analyses of karst springs, rječina spring and zvir, which ensure potable water for more than 200,000 people in rijeka county (croatia) methods: specific activities of selected radionuclides were determined by high resolution gamma spectrometric analysis and radiochemical separation method. values of hydrogen and stable isotope contents were determined by water equilibration method on isotope ratio mass spectrometer in conjunction with dual inlet and equilibration peripheral unit. results: anthropogenic radionuclides were detected in trace amounts. the results of the analysis show that the calculated yearly dose introduced to an adult human consuming 2 liters of water per day is approximately 20 sv. stable isotopes content of the spring waters indicates a dominant recharge of the analyzed hydrological system by winter precipitation. different isotopic variations of spring water as a consequence of sudden precipitation inputs in summer and autumn indicate that water discharged at rječina spring mainly originates from a big water reservoir situated in the wide mountainous region in the hinterland of the spring. conclusion: the examined water is potable and radiologically safe. stable isotope variations of the spring water show a fast reaction to sudden precipitation inputs, confirming the ecological vulnerability of karst springs. in light of recent heavy precipitation and flooding in different parts of croatia, a more systematic research on isotopic water composition should be encouraged. (mance d, lenac d, rubinić j. isotope studies of karst springs included in the water supply system of the city of rijeka (croatia). seemedj 2017; 1(2); 46-54) seemedj 2017, vol 1, no. 2 isotope studies of karst springs included in the water supply system… 47 southeastern european medical journal, 2017; 1(2) introduction rječina spring (rj) and zvir (zv) are the most important springs of the water supply system ensuring potable water for the city of rijeka and its surroundings. these are springs with relatively similar annual average discharges but different flow dynamics. rj springs at 325 m above sea level, has a mean annual output discharge of 7.38 m3s-1 and regularly dries out for several months a year. namely, rj is a seasonal spring that functions as an overflow for high and medium groundwater discharges (1). it provides high quality water for the city of rijeka and neighboring settlements through most of the year and its protection is of the highest priority. rj average water supply rate measures 0.69 m3s-1. zv springs at 3 m above sea level, has a mean annual distribution of 5.2 m3s-1 and a nominal minimum discharge of 1.5 m3s-1. the pumping from zv is activated, i.e. water from zv is used for water supply, when the rj discharge is reduced to 0.29 m3s-1 or it dries out completely (2). the two springs are under regular quality and health control checks by the water supply company and the teaching institute of public health of primorje & gorski kotar county (3), and have been subjects of many scientific investigations (1, 4). nevertheless, isotopic studies of these spring waters are not as frequent. the need for better understanding of complex processes taking place in this ecologically and socially important karst area is often emphasized (5). in order to expand the knowledge about rj and zv, we present the results of measurement for selected radionuclides in rj and zv water, as well as the results of monitoring hydrogen (2h) and oxygen (18o) stable isotope variations in rj water as a reaction to precipitation input events. to the best of the authors’ knowledge, there have not been any reports about rj and zv from the radiological point of view. the first report about the results of a systematic analysis of a stable isotope composition of the springs was given by mance et al. (6) and, before that, there were only sporadic reports (7, 8). the results presented here relate to short-term data collection, i.e. sampling during particular storm events, including observations related to rainfall inputs and stable isotope composition variations related to changes in rj discharge. the results of this type of studies can contribute to identifying water storage processes and mechanisms of groundwater recharge and discharge in the karst aquifer (9). they are also important for public health as they reveal information about the reaction time of karst springs to potential pollutants. materials and methods study area rj and zv are the largest springs of the rječina river (rr) basin, a typical dinaric karst basin. the basin is located in the dinarides of western croatia and southern slovenia (figure 1a). the rr basin covers approximately 500 km2 and has its main recharge area in the mountains of gorski kotar (croatia) and the snežnik massif (slovenia). the previous isotopic analysis found substantial evidence in favor of the conjecture that rj and zv are mainly recharged from the same average altitude and/or share the same groundwater reserves (6). the main lithological sedimentary units of the region are carbonate rocks, flysch and quaternary clastic deposits (figure 1b). according to the köppen-geiger climate classification (10), the associated climate type for the study area changes from cfa on the coast to cfb in hinterland. these are temperate climates, with precipitation regimen without regular dry seasons, and with hot (cfa) and warm summers (cfb). the mountainous part of the study area has an annual rainfall greater than 3000 mm, making it one of the rainiest regions in croatia (11). measurement methods and sampling for the purpose of this study, spring water samples were collected at rj and zv, while seemedj 2017, vol 1, no. 2 isotope studies of karst springs included in the water supply system… 48 southeastern european medical journal, 2017; 1(2) precipitation samples were collected at gumance (gum), the location that belongs to the recharge area of rj and zv basin. precipitation sampling site can be seen on figure 1b, while its coordinates and altitude are indicated in table 1. specific activity concentrations for natural (40k, 210pb, 226ra, 228ra, 232th, 235u, 238u) and anthropogenic (134cs, 137cs, 241am) radionuclides have been determined for two samples collected in march 2012 at springs rj and zv. activity concentrations were determined by high resolution gamma spectrometric analysis. additionally, for the determination of 226ra activity concentration, the radiochemical separation method was also used (12). measurements of activity concentrations were performed at the institute for medical research and occupational health (zagreb, croatia). effective dose calculation and results analysis were performed according to the regulation of croatian ministry of health and recommendations of the world health organization (who) (13, 14). the annual effective dose e (msv/year) resulting from water intake was estimated as (14): 𝐸 = 𝑞 ∙ ∑ 𝐶𝑖 ∙ ℎ𝑖 𝑖 where: q annual ingested volume of drinking water, assumed to be 0.73 (m3/year), ci the activity concentration of radionuclide i (bq/m3), hi dose coefficient for ingestion of radionuclide i by adults (msv/bq). drinking water is considered to be safe from the radiological point of view if the annual dose calculated based on the presence of radioactive isotopes in water, under the assumption of an average daily consumption of 2 liters, is below 0.1 msv (14). since there are no strict upper limits for activity of individual radionuclides in drinking figure 1. a) map of croatia showing the study area (circle); b) sampling locations and distribution of the main lithological structures in the study area. rj – rječina spring; zv – zvir; gum – gumance table 1. coordinates and altitudes (in m above sea level) of precipitation sampling site precipitation sampling site coordinates altitude (m a.s.l.) gumance (gum) n 45⁰ 28' 4'' e 14⁰ 24' 27'' 688 seemedj 2017, vol 1, no. 2 isotope studies of karst springs included in the water supply system… 49 southeastern european medical journal, 2017; 1(2) water, screening for gross and gross  radiation activity was also carried out (14). in order to investigate the karst aquifer discharge response to sudden precipitation inputs, daily sampling during storm events at spring rj was organized and precipitation samples were taken on gum. samples were taken during two storm events in late summer of 2013, followed by storm event sampling in the fall of 2013. on those occasions, both rain water (n = 10) and groundwater samples (n = 30) were collected. stable isotope contents of collected samples were measured by the water equilibration technique in stable isotope laboratory at the physics department of the faculty of medicine at university of rijeka. for the measurement, an isotope ratio mass spectrometer (deltaplus xp; thermo finnigan, germany) coupled with equilibration unit and dual inlet system was used. results were reported in -values (‰) on vsmow scale (15). the measurement precision was < 1 ‰ for hydrogen (2h), and < 0.1 ‰ for oxygen (18o). globally, for natural waters that are not under the influence of evaporation, the linear relationship between 2h and 18o is represented by the global meteoric water line (gmwl): δ2h = 8 · δ18o + 10 ‰ (16). there are local deviations from the gmwl, due to various different factors such as altitude, proximity to the sea, etc., which required the measurement of local meteoric water lines. one of these local meteoric water lines is the western mediterranean meteoric water line (wmmwl): 2h = 8 ∙ 18o + 13.7 ‰ (17). stable isotopes can be used as “fingerprints” to detect geographical origins of natural waters (18, 19). one of the ways to do this is by using d-excess, the hydrological parameter introduced by dansgaard (20): dexcess = δ2h – 8 ∙ δ18o. precipitation that originates from the atlantic ocean typically has d-excess values ≈ 10 ‰ and precipitation originating from the mediterranean has d-excess values > 15 ‰ (21). results gamma spectrometric analysis proved the existence of natural as well as anthropogenic radionuclides in examined water samples. the latter were only found in trace amounts. results of the analysis, i.e. activity concentration for radionuclides of interest, are presented in table 2. activity concentration of 226ra has been determined by using both gamma spectrometric method and radiochemical separation method, and results of these two methods do not differ significantly (table 2). calculated gross radiation activity for rj is 2.831 ∙ 102 bq / m3, and for zv it is 2.712 ∙ 102 bq / m3, while gross  radiation activity for rj is 2.509 ∙ 102 bq / m3 and for zv it is 2.443 ∙ 102 bq / m3. estimated effective dose for an adult whose daily intake is 2 l of water, for rj is 18 sv / year and for zv 23 sv / year. as it can be seen in figure 2, 2h and 18o values of spring water correspond to wmmwl, while the majority of precipitation samples lie between gmwl and wmmwl. figure 2 also shows spring water values corresponding to the lower part of value cluster of precipitation samples. d-excess values of precipitation in our study range from a minimum of 6.98 ‰ to a maximum table 2. results of gamma spectroscopic analysis of water samples radionuclide activity concentration (bq / m3) rječina spring zvir 40k 42.2 + 2.5 44.7 + 3.4 134cs < 1.2 + 0.2 4.4 + 1.5 137cs < 1.1 + 0.2 < 1.4 + 0.4 210pb 17.2 + 4.2 32.5 + 7.5 226ra () < 25.6 + 7.1 < 12.7 + 4.2 226ra () 21.1 + 6.3 5.4 + 2.8 228ra 3.4 + 0.5 2.6 + 0.7 232th 3.4 + 0.5 2.6 + 0.7 235u < 1.6 + 0.8 < 2.0 + 1.0 238u < 31.5 + 5.3 30.2 + 7.4 241am < 3.5 + 0.8 < 4.6 + 1.1 seemedj 2017, vol 1, no. 2 isotope studies of karst springs included in the water supply system… 50 southeastern european medical journal, 2017; 1(2) unlike the precipitation samples, standard figure 2. δ2h versus δ18o correlation diagram of rječina spring (rj) samples and precipitation samples collected at gumance (gum). global meteoric water line (gmwl) and west mediterranean meteoric water line (wmmwl) are shown for comparison. figure 3. deuterium excess values of rječina spring (rj) samples and precipitation samples collected at gumance (gum). the mean analytical uncertainty (+ 1.04 ‰) was as indicated by the error bars. seemedj 2017, vol 1, no. 2 isotope studies of karst springs included in the water supply system… 51 southeastern european medical journal, 2017; 1(2) deviation of 1.45 of 20.09 ‰ (figure 3). other precipitation d-excess values fluctuate about a mean value of 13.87 ‰, with standard deviation of 1.45 ‰. ‰. unlike the precipitation samples, dexcess values of spring water are all within the measurement error with a mean value of 15.13 ‰ and a standard deviation of 0.49 ‰. 18o time series of rj spring water collected in september 2013 show shifts towards more positive values that coincide with an increase of rj discharges (figure 4). the first such shift occurred on 18th september (rj, 18o = -7.71 ‰), although rain water collected on 17th of september had a lower value (gum, 18o = -9.2 ‰) than values of rj water sampled prior to that rain event (≈ -8.0 ‰ to -8.3 ‰). next significant rain event occurred between the 28th and 29th september 2013, with 62.3 mm of rain and 18o = -6.57 ‰. rj sample collected on the 28th september had the value of -7.53 ‰, although the discharge was very low. the discharge reached the highest value on 30th september, with corresponding 18o = -7.61 ‰. during the following days, both discharge rates and 18o values continued to decrease (figure 4a). after the described events, rj18o values oscillations were not significant any more, although there were major precipitation events in october 2013. the two most important of those precipitation events took place on 12th and 27th october, with precipitation amounts of 192.6 mm and 132.7 mm, respectively. 18o value for the first event was -7.14 ‰, and for the second one -7.81 ‰ (figure 4b), but a reaction to these events could not be detected in isotopic values of rj (figure 4a). figure 4. a) daily discharge and 18o groundwater series for samples collected at rječina spring; b) precipitation amounts and corresponding 18o values of samples collected at gumance (gum). the mean analytical uncertainty (+ 0.1 ‰) is indicated by the error bars. seemedj 2017, vol 1, no. 2 isotope studies of karst springs included in the water supply system… 52 southeastern european medical journal, 2017; 1(2) discussion values of radionuclide activity in spring waters of rj and zv reported here are well below guidance levels for radionuclides in drinking water recommended by who (14). the calculated yearly effective doses, obtained by using the concentration values of radionuclide activity in spring waters of rj and zv, assuming that the water is introduced into the human body on a daily basis, are ≈ 20 μsv. this is significantly below the acceptable limit of a dose (13). the analyzed rj and zv samples are not radiologically contaminated and water may thus be considered radiologically safe and used for drinking. mance at al. (6) showed that rj is under the influence of different hydrological conditions in comparison to other springs in the area. in order to provide some new, additional information about this complex system, samples of gum precipitation and rj groundwater were taken in late september 2013 and in the fall of 2013. both, an isotopic values correlation diagram (figure 2) and d-excess values of the spring samples (figure 3) indicate a mediterranean origin of the analyzed water, confirming the dominant recharge of the system by winter precipitation (6). an analysis of stable isotope dynamics shows a different composition of stable isotopes in groundwater during the three storm events. the first september storm event occurred after a relatively long period without significant precipitation. this event is difficult to comment given that a shift towards a more positive groundwater δ18o value occurred one day prior to the rain event on gum, and this rainfall had the second most negative δ18o value of all rain samples collected during this study (figure 4). a possible explanation is that a rain event that triggered the groundwater shift toward more positive δ18o value had happened earlier than the one on gum and that it had taken place deeper in the mountainous hinterland of the system, most probably on the slovenian snežnik massif. the second september event had significantly less negative δ18o precipitation values in comparison to rj values collected previously during the month (with the exception of the value that corresponded to the highest rj discharge of the previous event). as it already happened during the previous event, a shift towards less negative groundwater values occurred again a day prior to the event on gum (figure 4). once more, this suggests that an earlier event, taking place further in the catchment area, had activated the system prior to the event on gum. also, this indicates a fast reaction of the hydrological system to sudden precipitation inputs and a short retention time of newly infiltrated water in the underground. this is very important from the environmental protection point of view as it indicates an area highly susceptible to contamination. the introduction of a pollutant to the system would probably cause an immediate pollution of drinking water. stable isotope composition of groundwater samples collected after the third storm event was without significant oscillations although the precipitation amount was considerable and its δ values were less negative than those for groundwater sampled prior to the storm. bearing in mind that the system may be described by a dual porosity model (6), this situation might suggest that september storm events triggered the passage of groundwater through short and wide karst channels, while well mixed groundwater discharged in october originated from groundwater reservoirs. accordingly, it can be concluded that there is a scant amount of groundwater in the vicinity of rj, and the water discharged at rj mainly originates from a large water reservoir located in the mountainous region in the hinterland of the spring. the last precipitation event in this study occurred on 27th october, but the isotopic value of the collected precipitation was too close to the value of previously collected groundwater (figure 4), so this event could not be used for further analysis. seemedj 2017, vol 1, no. 2 isotope studies of karst springs included in the water supply system… 53 southeastern european medical journal, 2017; 1(2) although we got new insights in the functioning of the system, such as the one indicating that precipitation from the hinterland has greater influence on rj than precipitation occurring closer to it, there are still many unresolved questions. the reaction of the system to the precipitation inputs proved to be very fast, and daily sampling was probably not frequent enough to give a complete picture about the hydrological conditions. more frequent sampling, for example on an hourly basis, could give a better insight into the situation (9). conclusions the authors present the results of radioactive and stable isotopes analyses for rj and zv, the two largest and most important water supply springs of the city of rijeka, as well as precipitation samples collected at the assumed recharge areas of the two springs. the purpose of the paper is to provide a radiological analysis of their water quality and to expand the sources of available data for stable isotopes. measurements of radionuclides showed that the water is safe for human consumption. stable isotope data indicates a rapid reaction of the system to sudden precipitation inputs, thus confirming vulnerability of karst springs to potential pollution. we can confirm that the main water reservoir of the system is situated in the mountainous region of the basin, and that in the vicinity of the rječina spring there are only scant amounts of groundwater. the authors conclude that isotopic investigations of the spring water and precipitation collected on the assumed recharge area reveal some useful information on the hydrological system. recently, there have been catastrophic floods in different parts of croatia. we may treat the floods as indicators of a lack of detailed knowledge about the hydrological conditions in corresponding aquifers. therefore, more systematic isotopic analyses are strongly encouraged. acknowledgements data on rječina spring discharges were provided by the national meteorological and hydrological service. funding: the paper was supported by the ministry of science, education and sport of croatia, the croatian academy of sciences and arts (project “proučavanje pražnjenja krškog vodonosnika kao odgovora na ljetne olujne događaje” (studying karst aquifer discharge in response to summer storm events)) and the water supply company rijeka. competing interests: none to declare. references 1. biondić b, dukarić f, kuhta m, biondić r. hydrogeological exploration of the rjecina river spring in the dinaric karst. geol croat 1997;50(2):279-88. 2. rubinić j, sarić m. hidrologija vodnih resursa u slivu rječine. prošlost, sadašnjost i budućnost vodoopskrbe i odvodnje. 2005:199-207. 3. water supply company rijeka. analiza kvaliteta vode za piće. www.kdvik-rijeka.hr (28 august 2017) 4. frančišković-bilinski s, cuculić v, bilinski h, häusler h, stadler p. geochemical and stable isotopic variability within two rivers rising under the same mountain, but belonging to two distant watersheds. chemie der erde-geochem 2013;73(3):293308. 5. bonacci o, oštrić m, roje-bonacci t. prilog hidrologiji krškog izvora rječine. hrvatske vode 2017;25(100):99-108. 6. mance d, hunjak t, lenac d, rubinić j, roller-lutz z. stable isotope analysis of the karst hydrological systems in the bay of kvarner (croatia). appl radiat isot 2014;90:23-34. 7. biondić b, prestor j, biondić r, lapanje a, kapelj s, janža m, rikanovič r, urbanc j, singer d. transboundary aquifers between seemedj 2017, vol 1, no. 2 isotope studies of karst springs included in the water supply system… 54 southeastern european medical journal, 2017; 1(2) slovenia and croatia-the area between gulf of kvarner and gulf of trieste. geologija 2002;45/2:311-18. 8. hertelendi e, svingor é, futó i, szántó z, rank d. isotope investigation of lake vrana and springs in the kvarner area. rapid commun mass spectrom 1997;11(6):651-5. 9. trček b. epikarst zone and the karst aquifer behaviour: a case study of the hubelj catchment, slovenia. geološki zavod slovenije; 2003. 10. peel mc, finlayson bl, mcmahon ta. updated world map of the köppen-geiger climate classification. hydrol earth syst sci disscus 2007;4(2):439-73. 11. gajić-čapka m, perčec tadić m, patarčić m. digitalna godišnja oborinska karta hrvatske. hrvatski meteorološki časopis 2003;38(38):21-33. 12. bituh t, marovic g, petrinec b, sencar j, franulovic i. natural radioactivity of 226ra and 228ra in thermal and mineral waters in croatia. radiat prot dosimetr 2009;133(2):119-23. 13. narodne novine. pravilnik o granicama izlaganja ionizirajućem zračenju te o uvjetima izlaganja u posebnim okolnostima i za provedbe intervencija u izvanrednom događaju, http://narodnenovine.nn.hr/clanci/sluzbeni/2006_11_125_ 2771.html (15 august 2017) 14. world health organisation. guidelines for drinking-water quality. http://apps.who.int/iris/bitstream/10665/ 44584/1/9789241548151_eng.pdf (20 august 2017) 15. werner ra, brand wa. referencing strategies and techniques in stable isotope ratio analysis. rapid commun mass spectrom 2001;15(7):501-19. 16. craig h. isotopic variations in meteoric waters. science 1961;133(3465):1702-3. 17. celle‐jeanton h, travi y, blavoux b. isotopic typology of the precipitation in the western mediterranean region at three different time scales. geophys res lett 2001;28(7):1215-8. 18. brenčič m, kononova nk, vreča p. relation between isotopic composition of precipitation and atmospheric circulation patterns. j hydrol 2015;529:1422. 19. trček b, leis a. overview of isotopic investigations of groundwaters in a fractured aquifer system near rogaška slatina, slovenia. geologija 2017; 60/1:4960. 20. dansgaard w. stable isotopes in precipitation. tellus 1964;16(4):436-68. 21. julian j, araguas l, rozanski k, benavente j, cardenal j, hidalgo mc, garcia‐lopez s, martinez‐garrido jc, moral f, olias m. sources of precipitation over south‐eastern spain and groundwater recharge. an isotopic study. tellus b 1992;44(3):226-36. seemedj 2019, vol 3, no. 1 trefoil factor family 69 southeastern european medical journal, 2019; 3(1) review article trefoil factor family (tff): peptides with numerous functions 1 nataša kozina 1, ivana jukić 1 1 department of physiology and immunology, faculty of medicine, josip juraj strossmayer university of osijek, osijek, croatia corresponding author: ivana jukić, igrizelj@mefos.hr received: april 3, 2019; revised version accepted: may 27, 2019; published: may 31, 2019 keywords: trefoil factor family (tff), mucus, metabolic regulation, tff3-/-mice abstract the trefoil factor family (tff) consists of a group of small peptides and is highly expressed in tissue that contain mucus-producing cells, predominantly in the mucosa that lines the gastrointestinal tract. those peptides, which are highly important for epithelial restitution, may act in ways other than using the usual factors responsible for restitution. it was observed that several mechanisms are involved in the tffs’ promotion of restitution. in addition to that, peptides have other functions as well, e.g. they interact with the immune system. although the tffs’ therapeutic effects have been studied, it is uncertain which of the tffs’ in vitro properties are directly involved when it comes to their in vivo engagement. observing mice with genetic deletion of tff peptides can help us discover the function of the peptides that could be indicated by the deletion of the target protein or by adaptive regulation of some other protein that is affected by the deleted gene product. at the very least, a subset of functional networks controlled by a tff isoform and its downstream effectors can be identified by observing such mice. the discoveries related to the signaling mechanisms of the tff family leave much to discover about the distinct and shared pathways among those protective peptides. (kozina n, jukić i. trefoil factor family (tff): peptides with numerous functions. seemedj 2019; 3(1); 69-77) seemedj 2019, vol 3, no. 1 trefoil factor family 70 southeastern european medical journal, 2019; 3(1) biological properties of tff the trefoil factor family (tff) includes a group of small peptides, the first member of which was discovered about thirty years ago (1). tff1 (formerly known as breast cancer-associated peptide ps2), tff2 (formerly spasmolytic polypeptide sp) and tff3 (formerly intestinal trefoil factor) are three members of the trefoil factor family (tff) known in mammals. these three proteins are small compact peptides that have one or two trefoil domains. while tff1 and tff3 contain only one trefoil domain, tff2 contains two trefoil domains. the basic elements of a trefoil domain are 42–43 aminoacid residues. six cysteine residues form three disulfide bonds, creating a characteristic threeleafed structure (2). there are several studies that suggest that tffs can be regulated by cytokines and transcription factors (especially nf-kb) related to the immune system and that tffs can regulate them in return, but there is also data suggesting otherwise (3-6). tffs have been extensively studied in vivo and in vitro, with most data suggesting that these small peptides improve epithelial repair in the gastrointestinal (gi) tract and other body systems (7). tff’s role in mucosal protection and repair due to their compact structure, tffs are relatively resistant to proteolytic degradation in the stomach and small intestine (8). tffs can mostly be found as secreted molecules in the mucus covering normal epithelium. the predominant site of tff synthesis is the mucinproducing or goblet cells dispersed in epithelia. all three tffs are expressed in the stomach, being localized to the surface gastric mucosal cells (9). their mrna has been found in the brain, lungs, trachea, thyroid gland, salivary glands, prostate, uterus and other organs (10). the genes that code them are located on chromosome 21 (11). despite the fact that they have been found in almost all tissue containing mucus-secreting cells, tffs are predominantly expressed in the gastrointestinal tract. considering their appearance in mucosal tissue, it can be concluded that their functioning might be related to that of mucins. nevertheless, tff2 expression is not so common and there is a possibility that different tffs have different roles in the protection of the epithelium, which is corroborated by the complementary expression of tffs in the gi tract and by the simultaneous occurrence of each of them with their unique mucin type (muc); tff1 appears with muc5ac, tff2 with muc6, and tff3 with muc2 (12-14), although gastric and ocular co-localization of tff3 with muc5ac also occurs (15, 16). it is speculated that mucosal defense is improved by direct interaction of tff peptides and mucins. the role of tff peptides in cell migration was observed in several studies (17-20), predominantly as a consequence of the response of a damaged epithelium that strives to restore its continuity. in cases of small discontinuity of the epithelium, where cell proliferation is not required, restitution of the epithelium takes place soon after the injury, with coordination of the removal of damaged cells and the migration of healthy epithelial cells into the injured location (figure 1). the importance of efficient restitution from the physiological viewpoint is high, as the loss of fluids and electrolytes has to be stopped and the luminal antigens and bacteria have to be prevented from entering the tissue and immune cells of the host. proliferation occurs instead of restitution when tissue is more severely damaged. seemedj 2019, vol 3, no. 1 trefoil factor family 71 southeastern european medical journal, 2019; 3(1) figure 1. the role of tff peptides in epithelial restitution tff and energy metabolism the influence of trefoil factor proteins on energy metabolism can be observed in mice with tff3 gene knockout (tff3-/mice). the tff3 knockout mice have a different expression of mirna associated with the glycolysis and gluconeogenesis metabolic pathways compared to wild-type mice. the tff3 knockout mice have a significantly lower body weight compared to the wild type (21). a change in the body mass of mice did not occur in the research with increased expression of the tff3 gene (22). the fatty changes in the liver of mice have been connected with the change of expression of the tff3 gene (23). research has shown that the tff3 protein participates in glucose metabolism. similarly, a study showed that hepatic tff3 expression levels were lower in obese (ob/ob) and high-fat-diet-induced obese mice. cellular glucose output in mice decreased as a consequence of overexpression of tff3 in primary mouse hepatocytes, which inhibited the expression of gluconeogenic genes. experiments using the glucose tolerance test and insulin tolerance test showed that adenovirus-mediated overexpression of tff3 in diabetic or obese mice improved glucose tolerance and insulin sensitivity. the results also showed that tff3 peptides are a factor in glucose homeostasis and insulin sensitivity. consequently, it was concluded that said peptide might be a part of successful modern therapies aimed at metabolic disorders related to type 2 diabetes mellitus (24). increasing concentrations of glucose and insulin treatment boosted the expression of tff3 in intestinal epithelial cells. in addition to that, insulin treatment caused the upregulation of human sodium/glucose cotransporter 1 (hsglt1), which additionally increased intracellular glucose levels. downregulation of tff3 was observed in diabetes mellitus type 1 patients, but the values were modified by insulin treatment. it was discovered that insulin signaling was important for the optimal expression of tff3 in intestinal epithelial cells, as it elevates intracellular glucose levels and mediates gene expression (25). aberrant energy metabolism in the liver promotes insulin resistance, diabetes, and nonalcoholic fatty liver diseases (26). it was recently shown that liver triglyceride accumulation does not cause cellular injury in the liver; the primary causes of liver injury via increased oxidative stress are free fatty acids or their metabolites (27). changes in lipid metabolism, especially the increase of saturated fatty acids, are associated with increased endoplasmic reticulum (er) stress, oxidative stress and liver injury in the course of development of fatty liver disease (28). sirtuin 1 (sirt1) plays a key role in metabolic regulation, adaptation and oxidative stress. acting as a nuclear metabolic sensor and deacetylating a wide range of targets, it leads to epigenetic modifications of histones and modulation of transcription factors or metabolic enzymes (29). in addition to sirt1, peroxisome proliferatorseemedj 2019, vol 3, no. 1 trefoil factor family 72 southeastern european medical journal, 2019; 3(1) activated receptors (ppars) also have an important role in cell metabolism (30). in case of tff3 deficiency, the profile and accumulation of fatty acids (fas) in the liver are affected (table 1), with no obvious oxidative stress increase, although the expression/activity of monitored enzymes changes, as does the level of sirt1 and pparg protein. due to the strong downregulation of hepatic tff3 in diabetic/obese mice, its presence in circulation and its regulation by food/insulin, tff3 represents an interesting new candidate for research in metabolic relevant conditions (31). table 1. fatty acids in liver of tff3 -/ mice compared to wild type (elevated ↑, decreased ↓) fatty acids tff3 -/ saturated c14:0 myristic acid c18:0 stearic acid c20:0 arachidic acid monounsaturated c16:1 palmitoleic acid (ω-9) c18:1 oleic acid (ω-9) c18:1 vaccenic acid (ω-7) c20:1 eicosenoic (gondoic acid) (ω-9) polyunsaturated c20:2 eicosadienoic acid (ω-6) c20:4 (aa) arachidonic acid (ω-6) c18:3 (ala) alpha linolenic (ω-3) c22:6 (dha) docosahexaenoic (ω-3) ratio ω-3/ω-6 ↓ ↑ ↑ ↓ ↓ ↓ ↓ ↑ ↑ ↓ ↑ ↑ tff’s participation in defense against harmful agents another role of the tff3 protein is the defense of the organism against harmful agents. mice that cannot synthesize enough tff3 protein in their liver are deprived of the protective effect of the tff3 protein in the serum after myocardial (32) and brain ischemia, which consequentially leads to greater tissue damage. thus, in such mice, a significantly higher activity of caspase 3 and a higher level of cell death in the ischemic cerebral lesion were observed, together with a larger fraction of cerebral infarcts and a smaller fraction of injuries in the cerebral hemisphere, accompanied by more severe forelimb motor deficits. since the mice were tff3-deficient, recombinant tff3 was administered intravenously and it reversed changes in cerebral injury and forelimb motor function, pointing at the existence of an endocrine neuroprotective mechanism that uses tff3 from the liver in experimental cerebral ischemia/reperfusion injury (33). tff3-/mice have difficulties with regeneration of the mucous membrane of the gastrointestinal tract (34). high-salt diet (hs) causes endothelial dysfunction and vitiates vascular reactivity to various stimuli. in a recent study, transgenic tff3-/mice were introduced as a new model, characterized by a favorable ratio of ω-6/ω-3 free fatty acids and modified metabolism of arachidonic acid (aa). the results showed that acute hs intake has a much smaller impact on fir (flow-induced response) in tff3-/mice compared to the wild type (wt) (35). the study seemedj 2019, vol 3, no. 1 trefoil factor family 73 southeastern european medical journal, 2019; 3(1) showed that hs intake does not affect no production in tff3-/mice (36). according to another study, although the tff3 peptide is not expressed in an intact corneal epithelium, its expression is extensively upregulated following an epithelial injury. in addition to that, corneal injuries in tff3-/mice take much more time to re-epithelialize compared to similar injuries in wild-type mice. in case of alkali-induced corneal wounds, external application of recombinant tff3 to the wounds speeds up the in vivo and combined in vivo/in vitro model wound healing in both wild-type and tff3 /mice. this proves that tff3 has a key role in the mechanism of corneal wound healing, which opens a possibility of creating new ways of coping with non-healing wounds (37). tff in the respiratory system, pregnancy and tumorigenesis a study (38) describing a murine asthma model found that trans-differentiating clara cells specifically express tff1 which is stored in a specific subset of secretory granules. this is proof that tff1 is an autocrine factor for the trans-differentiation of clara cells into goblet cells. another study (39) showed that tffs play such a role in the differentiation of the airways as well, showing the induction of tff3 synthesis with the differentiation in in vivo humanized tracheal xenograft and in vitro air-liquid interface culture models. in addition to that, exogenous tff3 promoted differentiation of ciliated cells in an egf-receptor-dependent manner. both studies implied that tffs may have important roles in different processes of differentiation of airways, making them promising new targets in treatment of severe chronic and acute airway diseases. dynamic changes of trefoil factor proteins in a pregnant woman’s serum point at their importance in embryogenesis (40). the presence of the tff3 protein in the cartilage of mice fetuses during endochondral ossification has been identified, while the exclusion of the tff3 gene causes changes to the histomorphological structure of cancellous bone, as well as hearing disorders and accelerated presbycusis, which indicates that it has a role in morphogenesis of organs (41-43). the tff3 gene participates in the proliferation of pancreatic cells – decreased expression of the tff3 gene leads to decreased proliferation of -cells, while increased expression leads cells, having no influence on their function (44). the tff3 protein is also related to angiogenesis, which makes it an important factor in tumor pathogenesis (45). research has identified increased expression of the tff3 gene in gastrointestinal and lung tumors, advanced prostate cancer, hepatocellular carcinoma and other tumors (46-49). expression of the tff3 gene has a predictive role in breast tumors (50) and is simultaneously identified as a valuable and easily detected biomarker in screening for stomach cancers. moreover, serum tff3 might predict gastric cancer more efficiently than the pg test, while the combined testing of serum pg (pepsinogen test) and tff3 could make gastric cancer screening even more efficient (51). conclusions despite the fact that not much is known about the tff signaling pathways, some straightforward benefits of tff peptides for healthy and damaged tissue have been discovered. tffs are pivotal for mucosal protection and repair of epithelial surfaces, and they also have a role in cancer development and progression. trefoil factors can be used as prognostic markers for different types of carcinoma. however, their biological effects are still unknown. considering that there are not many studies on the influence of the tff peptides on vascular reactivity, it would be interesting to find out more about their role in it. acknowledgement this article is written in the frame of the project vif2018-mefos-09. seemedj 2019, vol 3, no. 1 trefoil factor family 74 southeastern european medical journal, 2019; 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22(5): 1251-9. 45. dhar dk, wang tc, tabara h, tonomoto y, maruyama r, tachibana m, kubota h, nagasue n. expression of trefoil factor family members correlates with patient prognosis and neoangiogenesis. clin cancer res. 2005;11(18):6472-8. 46. im s, yoo c, jung jh, choi hj, yoo j, kang cs. reduced expression of tff1 and increased expression of tff3 in gastric cancer: correlation with clinicopathological parameters and prognosis. int j med sci. 2013;10(2):133-40. 47. qu y, yang y, ma d, xiao w. increased trefoil factor 3 levels in the serum of patients with three major histological subtypes of lung cancer. oncol rep 2012;27(4):1277 83. 48. vestergaard em, borre m, poulsen ss, nexø e, tørring n. plasma levels of trefoil factors are increased in patients with advanced prostate cancer. clin cancer res 2006;12: 80712. 49. okada h, kimura mt, tan d, fujiwara k, igarashi j, makuuchi m, hui am, tsurumaru m, nagase h. frequent trefoil factor 3 (tff3) overexpression and promoter hypomethylation in mouse and human hepatocellular carcinomas. int j oncol 2005; 26(2): 369-77. 50. may fe, westley br. tff3 is a valuable predictive biomarker of endocrine response in metastatic breast cancer. endocr relat cancer 2015; 22(3): 465-79. 51. huang z, zhang x, lu h, wu l, wang d, zhang q, ding h. serum trefoil factor 3 is a promising non-invasive biomarker for gastric cancer screening: a monocentric cohort study in china. bmc gastroenterology 2014; 14:74. seemedj 2018, vol 2, no. 1 a partner relationship in meeting the needs 49 southeastern european medical journal, 2018; 2(1) a partner relationship in meeting the needs of patients and their families 1 brankica juranić1, štefica mikšić1, metka lipič baligač1,2, klaudia knezić1,3 1 faculty of dental medicine and health, josip juraj strossmayer university of osijek, croatia 2 general hospital murska sobota, murska sobota, slovenija 3 university medical centre zagreb, zagreb, croatia corresponding author: brankica juranić, juranicbrankica@gmail.com received: aug 28, 2017; revised version accepted: apr 5, 2018; published: november 27. 2018 keywords: patient, family, nurse, spouses, decision making abstract a nurse is the first person who comes in contact with patients and their family members. she shares information with them by verbal and non-verbal communication, with the goal of establishing a good relationship and focusing her attention on the patient as a whole person. nurse’s sincere thoughts and feelings create a sense of security and open communication, which are key elements for providing healthcare and inclusion of the family in the decision-making process. a nurse who spends her time by the patient’s side plans, implements, evaluates and documents the changes in the patient’s condition. that way the healthcare is focused on meeting the physiological needs and maintaining stable condition of the patient. struggle for life doesn’t leave any time for the family to adjust to the new situation. establishing the patient – family – nurse relationship is important not only in the beginning, but also during the whole treatment and decision-making process. when relationships within the family are stable, the family has an irreplaceable role in developing a sense of security and belonging for the patient. providing care for the patient is a priority for the healthcare team, whereas the most significant thing for the family are sincere and correct information about the patient’s current condition, the effect of the applied therapy and the possible outcome of the treatment. developing care philosophy focused on the family and the holistic approach includes assessment of the family’s needs and the impact of the disease on its overall functioning. cultural factors play a significant role in the ability to understand not only the patient’s but also the family’s point of view. medical, ethical, legal and a whole range of other problems connected to the receiver and provider of services can be avoided by effective communication. the purpose of this paper is to highlight the importance of a partner relationship, focusing on the family and the essential role of the nurse in decision-making. (juranić b, mikšić š, lipič baligač m, knezić k. a partner relationship in meeting the needs of patients and their families. seemedj 2018; 2(1); 49-56) seemedj 2018, vol 2, no. 1 a partner relationship in meeting the needs 50 southeastern european medical journal, 2018; 2(1) introduction providing quality healthcare to the patient, cooperating with the family and establishing a good relationship in a multidisciplinary team are requirements nurses face in a holistic approach to the patient. frequency and availability of information and discussion about the uncertainty of the prognosis help in creating a good working environment among healthcare team members and successful cooperation with the family. an illness is not stressful only for the suffering patient but also for family members and other persons from the patient’s environment with whom he/she is in contact. the family’s primary role is to support and protect its member throughout the course of the illness and the related decision-making process. this process is complex, the family considers it to be the most important one, but more importantly, the quality of service provision and the patient’s quality of life depend on it. in deciding about procedures and interventions for the patient whose life is threatened, the recommended model is the joint decisionmaking, which includes clinicians, family members, relatives or a guardian, and it is important that all of them receive correct, valid and necessary information at the same time. responsibility and decision-making are ethical issues. the family wants to actively participate in order to fulfil the wishes of the patient and expects open and sincere communication. in practice, a positive effect of family support on the outcome of a serious illness requiring intensive care has been recognised for decades. the role and participation of the nurse in decision-making is connected to the quality of health services, and the nurse’s thoughtfulness and kindness towards the patient and the family contribute to the development of feelings of security and trust. through her work by the patient’s side and by providing comfortableness, gentle touch and information about any changes in the patient’s condition, the nurse helps the family members develop an understanding of the patient’s condition, as well as a perception of future development of the situation. by frequently following-up on the patient, the nurse becomes very well acquainted with the patient's personality. partnership approach and team members for the sake of cooperation within the team and a greater satisfaction of the patient and family members, members of a multidisciplinary team have to be responsible, properly trained, possess good communicational skills and be ready to hear other people and acknowledge their opinion. nurses’ skills imply more than just instrumental and technical skills and providing information to the patient they also pertain to integration of affective and relational aspects of communication. communication is the basis for ensuring satisfaction and understanding of expectations, it is to be conducted in detail, and often. family members need to be given an opportunity to express their concern and seek clarification for anything that they do not understand during the discussion, in order to be able to make right decisions (1). their needs and satisfaction depend on many factors, such as their social and economic situation, availability of health care or literacy. anxiety that appears when someone is facing death can be alleviated through existential care, during which healthcare workers explore their own sense and values after witnessing the suffering of a patient whose life is threatened by an illness (2). in terminal patients, the comfort is made by sharing fears with the patient and the family (2), who have many unanswered questions, depending on their unique cultural, economic and religious background. adequate and efficient communication among family members in the decision-making process protects the autonomy of the patient (3,4). in order for the nurses to be able to meet all the requirements in providing holistic care, they need to become actively involved in the discussion about the patient and in the actual decision-making process, which would result in greater satisfaction regarding communication with the family. it would also help alleviate seemedj 2018, vol 2, no. 1 a partner relationship in meeting the needs 51 southeastern european medical journal, 2018; 2(1) patients’ fear, and prevent burnout in nurses. it is necessary to implement continuous training in communicational skills and the nursing act needs to be amended to include duties and competences, counselling and support for the patients’ families and the development of a family-oriented concept (5,6). without allencompassing organizational dedication to the patient and the family, these challenges represent a barrier that prevents change of healthcare system culture. care for the patient and the family is focused solely on the service provided to the users (7). partnership approach acknowledges and attempts to use the knowledge and skills of both participants, and each brings a different, but potentially valuable and complementary relationship (8), which includes sharing ideas and mutual teaching (9). nurses and decision-making professionalism in nursing needs to reflect one’s enjoyment in work. nurses have to provide care by seeing the world through patients’ eyes and meet their needs while sharing the experience of being hospitalised, developing a mutual partner relationship. nurses have to be capable and willing to spend time with the patient and to be available to both the patient and their family. they should thoughtfully consider a person as an individual, not merely acquired compassion with the transfer of information in a non-verbal way, but also reacting to signs and expressing feelings of empathy. nurses share their concerns and develop a feeling that each patient is an individual by actively listening to the patients and not by treating them as a number. they encourage inner strength and hope for accepting and implementing the therapy, increase motivation and raise the quality of their relationships. nurses are not always able to correctly asses and meet patient’s needs (10), and they are often not included in the decision-making process (11,12). in their work, nurses encounter unsatisfactory communication and cooperation with doctors, which is a consequence of insufficient participation in the decision-making process and receiving information about the patient. nonparticipation in the decision-making and the lack of openness and dialogue arouses suspicion and distrust in the course and outcomes of the treatment, and deepens the feeling of anxiety and loneliness in family members in a time of a difficult existential situation. nurses who devote more time to patients and members of their families have valuable information about the patient and the situation within the family. the family can share their concerns regarding the patient with them, and the outcomes of good communication are associated with stress reduction. consequences of a lack of information are increased loneliness, insecurity and lack of understanding in the decision-making process. nurses need more education in developing communication skills required for their work with a dying patient and his/her family (13). in those circumstances, a lack of trust and communication stimulate fear and patients become overwhelmed with anxiety and anger (2). some nurses give patients and their families an opportunity to ask questions about issues that cause anxiety and hopelessness, or to talk about their feelings and desires for the future (2). when there is an increased likelihood that a patient will die, healthcare team members have to be ready to talk about it, because that is expected by the patient and his/her family. family and decision-making a research conducted by reesal bath (2000) came to a conclusion that relatives need information but cannot always receive it from health professionals. considering that they are in a state of physical and emotional exhaustion, it is unknown to what extent the patients are capable and competent to participate in the decision-making process, hence the responsibility is perceived as divided among those who are included in such process (14). the lack of involvement of the family and lack of information, openness and reciprocity in decision-making leads to feelings of abandonment, inability to protect or support. a passive attitude in the process of decisionmaking can lead to anxiety and depression (15), whereas active involvement result in traumatic seemedj 2018, vol 2, no. 1 a partner relationship in meeting the needs 52 southeastern european medical journal, 2018; 2(1) stress symptoms (16). interaction between the family, nurses and doctors is of vital importance, where mutual trust is crucial (17,18). a systematic approach enables expertise, communication skills, awareness, empathy and ability to adjust the plan to the relevant situation (19), which enables good cooperation with the family. the family needs to actively participate, plan and jointly make decisions regarding care provision to their loved-ones. caregivers are faced with challenges regarding communication with the patients and providing information on their condition. the patient needs to choose to whom the information relating to his/her health can be provided. providing information is a matter of privacy and wish which should be of utmost importance (20). access to information about the patient’s health condition, requirements and quality of relationship with the staff are the primary needs of the family, and meeting those needs is a primary responsibility of doctors and nurses in intensive care units, which is important for assessment of care quality (21). the family finds itself in a whirlpool of insecurity, shock, helplessness and confusion. the support is priceless (21). little research has been conducted on interventions with families of critically ill patients, and almost no one has done anything to improve communication between the healthcare team and the patient’s family (9). nurses have a leading role in facilitating cooperation between the family physician, nurses, family members, applying a collaborative approach to the problem through planning and providing holistic care and integrating palliative care. communication needs to be adjusted to suit each family individually. monitoring of the protocol and application of standardized procedures in practice would help with the assessment for solving the patient’s and the family’s problems. relatives of patients with carcinoma have different needs; however, their priority is quality care, in which they include their own perception and seek nurses’ support. relatives, in turn, provide immense support in reduction of stress and anxiety, and it is therefore important that the patient, while still able to do so, nominate a person who is to be informed about his/her condition. the family and the patient need time to realise that futile life-sustaining procedures are conduct to give time to the family to prepare itself for the final decision step by step (22-26). in such a way, providing care to the patient and caring for the family make up a unit in the process of joint decision-making (24). communication in the er unexpected admission of a patient in the er for the purposes of reanimation is a traumatic experience for the patient and his family, and it requires great support from the medical staff during and after the resuscitation. important features of care for the family are presence and proximity of loved-one, meaningful information, support provided by the staff and comfort. after repeated but unsuccesful resuscitation, those who suddenly find themselves in mourning feel the need to see the body of their loved-ones and touch them, which enables them to better come to terms with their loss and ultimately helps them in the mourning process.. in such situations they want to hear (and ask for) an explanation regarding the circumstances of their lovedone’s death but while they are in the state of shock, they cannot take in verbal information. many authors also describe a psychological trauma experienced by the family members who were there when the resuscitation took place. in the aspect of healthcare providing, nurses meet basic human needs and consider that it is not possible to provide genuine holistic care in these situations. they often exhibit their inability to do so through insensitivity, disinterest, inhumanity and coldness (27). determining the ability to make decisions on behalf of the weakened and exhausted patients depends on the assessment of the kind of help that caregivers can provide. the family is uncertain, they do not know what the patient knows or does not know, or what the patient him/herself would give their consent to or what they would be able to give their consent to. they often experience moments of hope related to improvement, but they also experience constant fear of deterioration or possible loss of loved-one, and confrontation with termination of treatment and acceptance of reality. relatives suffer from mental disorders such as fear, depression, uncertainty, seemedj 2018, vol 2, no. 1 a partner relationship in meeting the needs 53 southeastern european medical journal, 2018; 2(1) helplessness and hopelessness, but they also struggle with financial difficulties, problems with their work or education, problems with the changing of roles in the family and social isolation. they often suffer from headache, back pain, sleeping disorders, fatigue, loss of appetite and reduction in physical strength (28) and they experience a high level of emotional stress (29,30). role of the partner in providing support partners, relatives and close friends are not just advisors, but also a main source of support for patients with carcinoma (31) through provision of emotional stability and help during the treatment and in the patients’ everyday functioning. for women suffering from breast cancer, partner’s emotional support is of great significance and it alleviates their suffering. not only does this feeling reduce stress, it also enhances the quality of life, boosts the selfesteem of these women and strengthens their trust in partner (32). research has shown that in a situation in which one of the partners gets sick, it is the patient’s partner who carries greater mental load than the patient him/her self (31), and their involvement in the decision-making process is the best way of providing support. illness of a family member influences the balance in such a way that it comes to destabilisation and loss of security for all family members. they are faced with requirements caused by the disease, such as providing increased emotional support for the sick partner, redistributing life plans and taking on new tasks or roles within the family (33). patients’ partners seek and must receive emotional support in order to be able to carry their own burden and return to normal function. specificity of communication with elderly patients in an individual approach and contact with an elderly patient, a nurse needs to be familiar with what the patient believes, wants and whether he/she has any specific needs (34). the nurse encourages the patient not to lose hope, to accept the changes and to be persistent in order to achieve greater independence, (35) and also helps the patient in achieving his/her goals. needs are changeable and dynamic, and in order to meet them, the nurse is expected to provide support as care coordinator (36). life expectations of elderly patients are evergrowing, and conducting rehabilitation is a key factor in ensuring their independence and improvement of life quality after a traumatic event, in facing deterioration caused by a chronic health condition or in their preoccupation with the feeling of losing themselves (37). developing a relationship of trust helps the patient in carrying out activities which he/she is capable of performing, in a right manner and in an environment in which he/she feels safe and has a sense of existence. a nurse recognises that through his/her knowledge and intuition, he/she has an irreplaceable role in assessing and providing motivation and support, encouraging and boosting of self-esteem, educating on self-care and helping in everyday activities, using different aids or demonstrating to the patients how to do a certain activity in a simpler way. intuitive understanding of patients and their family members is a concept that nurses have recognized as a road towards better outcomes (38). family members feel fulfilled and invest great effort in order for their lovedone to get the best possible care. patients sometimes start feeling as if they are dependent on nurses, whose role is not sufficiently recognized. in order to achieve greater independence and progress, the method of providing rehabilitation is equally important as all the other interventions and procedures. research has shown that some family members perceive a nurse based on their own experiences and stereotypes, and not based on the current situation, which does not contribute to the welfare of the patient and his/her motivation. conclusion nurses should invest more effort in order for the partner relationship to become better integrated in the standards of healthcare provision. the seemedj 2018, vol 2, no. 1 a partner relationship in meeting the needs 54 southeastern european medical journal, 2018; 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38(2):106-110. seemedj 2019, vol 3, no. 1 low selenium diet in male sprague-dawley rats’ serum 50 southeastern european medical journal, 2019; 3(1) original article effect of low selenium diet on glutathione peroxidase 3 concentration in male sprague-dawley rats’ serum 1 nikolina kolobaric 1, ana stupin 1, 2, petar susnjara 1, lidija baric 1, anita matic1 1 faculty of medicine osijek, josip juraj strossmayer university of osijek, osijek, croatia 2 faculty of dental medicine and health in osijek, josip juraj strossmayer university of osijek, croatia corresponding author: anita matic, anita.cosic@mefos.hr received: march 30, 2019; revised version accepted: may 7, 2019; published: may 31, 2019 keywords: antioxidative enzymes, glutathione peroxidase 3, se, sprague dawley rats abstract aim: determination of antioxidative enzyme glutathione peroxidase 3 (gpx3) serum concentrations after consumption of food which contains different concentrations of selenium (se). research subjects and methods: four-week-old sprague dawley rats consumed food containing different concentrations of se (food divan) over a period of 10 weeks. the animals were divided into two groups: 1) normal se (0.363 mg/kg se) and 2) low se (0.030 mg/kg se). each animal was weighed at the end of protocol, and serum samples were collected for determining gpx3 concentrations. all experimental procedures were in compliance with the european convention for the protection of vertebrate animals used for experimental and other scientific purposes and approved by the ethics committee of the faculty of medicine in osijek and the ministry of agriculture of the republic of croatia. results: different concentrations of se in food did not cause a change in body weight. food containing the recommended intake of se according to the guidelines of the world health organization significantly increased gpx3 enzyme concentration (13.96±0.42 mg/ml) when compared to low selective se (12.04 ± 0.33 mg/ml, p = 0.002). conclusion: serum concentration of the antioxidant enzyme gpx3 depends on the concentration of se in food. it is shown that, in comparison with food with low se levels, food containing a normal concentration of se is enriched with the antioxidant gpx3 which, according to numerous studies, has a protective role in the human body. (kolobaric n, stupin a, susnjara p, baric l, matic a. effect of low selenium diet on glutathione peroxidase 3 concentration in male sprague-dawley rats’ serum. seemedj 2019; 3(1); 50-59) seemedj 2019, vol 3, no. 1 low selenium diet in male sprague-dawley rats’ serum 51 southeastern european medical journal, 2019; 3(1) introduction the most important antioxidant enzymes are catalase, glutathione peroxidase, glutathione reductase and superoxide dismutase, which have harmful oxidative metabolic intermediates. these enzymes require cofactors selenium (se), zinc, copper, iron, and magnesium for their catalytic activity (1). glutathione peroxidase (gpx), which is the subject of our study, is a family of enzymes that metabolize hydrogen peroxide and lipid hydroperoxide into water. they have a binding site for se, which oxidizes in reaction with hydrogen peroxide (2-4). glutathione peroxidase is involved in protection against oxidative stress in that it participates in the transfer of amino acids through the plasma membrane, removes the hydroxyl radical and the singlet oxygen, thereby detoxifying the hydrogen peroxide and the lipid peroxide by the catalytic action of gpx. it can replenish the active forms of the most essential vitamins, i.e., vitamins c and e (5). it is known that oxidative stress is one of the pathogenic mechanisms that cause disorders of the vascular system and contribute to development and progression of various cardiometabolic diseases (hypertension, diabetes, atherosclerosis, obesity). a diet enriched with trace elements, which increase the concentration of antioxidant enzymes, could provide better protection against oxidative stress (6). as part of gpx, se is important for the regulation of the oxidative system (7-9). it is an essential trace element which must be ingested in sufficient quantity through food. in many countries, there is malnutrition due to the lack of se as a micronutrient. inadequate se intake causes many different disorders (necrotizing cardiomyopathy, peripheral myopathy, reduced muscle tone, concentration problems, hair loss and nail splitting) (10-12). in our previous animal study, we showed that low dietary se content affects the function of aorta, reduces achinduced relaxation, which is dominantly mediated by no, and also increases the level of local oxidative stress (13). human studies have shown that se concentrations are inversely related to mortality and occurrence of cancer (14, 15). se functions as an antimutagenic agent that prevents the transformation of healthy cells into malignant ones; it is assumed that these protective effects are primarily associated with the activity of gpx (16, 17). glutathione peroxidase 3 (gpx3) is the only enzyme in the gpx group that functions in extracellular space. the substrates for this enzyme are hydrogen peroxide and phospholipid hydroperoxides, which play a significant role in the antioxidative processes in the blood (18) in that they decrease oxidative stress by reducing h2o2 and organic hydroperoxides to their corresponding alcohols and oxygen (19). since increased gpx3 activity is found in certain subtypes of tumors, like ovarian tumors (20), it can also be used as a biomarker. this study aims to determine the level of the gpx3 antioxidative enzyme in serum samples with different levels of se and to determine whether se causes a change in body mass. materials and methods ethical approval experimental procedures complied with the european convention for the protection of vertebrate animals used for experimental and other scientific purposes (council of europe no 123, strasbourg 1986) and were approved by the ethics committee of the faculty of medicine, university of osijek (class: 602-04/14-08/06, no: 2158-61-07-14-05), and authorized by the ministry of agriculture of the republic of croatia (class: up/i-322-01/14-01/90, no: 525-10/025515-4). the tests were performed in the laboratory for vascular physiology and laboratory for molecular and clinical immunology at the department of physiology and immunology of the faculty of medicine osijek, croatia. seemedj 2019, vol 3, no. 1 low selenium diet in male sprague-dawley rats’ serum 52 southeastern european medical journal, 2019; 3(1) experimental groups food was prepared at the faculty of agrobiotechnical sciences osijek, croatia, based on the wheat briquettes recipe developed by mucedola, italy. wheat was specially grown in soil with different concentrations of selenium and zinc, so that the concentration of said micronutrients was adjusted according to the requirements of the experiment. four-week-old healthy male sprague dawley rats (n = 7 per group) consumed the above-mentioned food for ten weeks. the animals were randomly divided into groups: 1) normal se group (0.363 mg/kg se) and 2) low se group (0.030 mg/kg se). rats were housed doubly in shoebox-style cages with free access to food and tap water, housed in a temperature of -21˚c-22˚c, humidity and light-controlled room, maintained on a 12:12 hour light : dark cycle. determination of se (normal or low concentration) in food is consistent with previous studies (13, 21-24). after 10 weeks of feeding, the animals were weighed and then anesthetized using a combination of ketanest s 75 mg/kg (ketanest s 25 mg/ml, 2 ml ampoules, pfizer) and midazolam 0.5 mg/kg (midazolam torrex 5 mg/ml, 3 ml, torrex chiesi pharma). blood samples were collected immediately after decapitation (arterial and venous blood) in empty tubes without anticoagulants to obtain serum and centrifuged at 3500 rpm for 10 minutes. the separated serums were stored in a refrigerator at -80 ° c until analysis. glutathione peroxidase 3 (gpx3) determination in serum samples gpx3 concentrations were determined using the commercially available enzyme immunoassay kit purchased from lifespan biosciences, usa (lsbio cat. no. ls-f6289). each well of the supplied microtiter plate was precoated with an antibody. first, 100 μl of serum samples and standards (in duplicates) were put in their appropriate place on the elisa plate and incubated for 1 hour at 37 ° c. after the first incubation, the unbound standard or sample was washed away, 100 μl of biotin-conjugated detection antibody was added to each well and the same time and temperature incubation was continued. after that, it was washed 3 times and avidin-horseradish peroxidase (hrp) conjugate was added, followed by a 30-minute incubation at 37 ° c. a tmb substrate was then added, which reacted with the hrp enzyme, resulting in color development. at the end of the protocol, stop solution was added to terminate the color development reaction and optical density (od) was measured at a wavelength of 450 nm on the pr 3100 tsc microplate reader in the laboratory for molecular and clinical immunology at the department of physiology and immunology of the faculty of medicine, josip juraj strossmayer university of osijek. statistical analysis differences in normally distributed numerical variables between groups were tested with the student t-test, and in case of deviations from normal distribution with the mann-whitney u test (sigmaplot version 11.2, systat software, inc., chicago, usa) the level of significance was determined at p < 0.05. the sample size was determined using the sigma plot version 11.0 program. for the power of the test of 0.8, p value less than 0.05 and the minimum expected difference of 0.25, it was found that at least 4 animals per group were required. results are presented below as the mean ± standard deviation (sd). results effect of diet on body weight there was no difference in body weight between the normal se and the low se group (p < 0.05) (figure 1). seemedj 2019, vol 3, no. 1 low selenium diet in male sprague-dawley rats’ serum 53 southeastern european medical journal, 2019; 3(1) figure 1. body weight in experimental rat groups gpx3 serum concentration the group of animals that consumed food with normal se content had significantly increased gpx3 enzyme concentrations (13.96 ± 1.11 ng/ml) compared to the low selective se content group (12.04 ± 0.89 ng/ml, p = 0.002) (figure 2). figure 2. concentration of the antioxidative enzyme gpx3 (ng/ml) in serum samples seemedj 2019, vol 3, no. 1 low selenium diet in male sprague-dawley rats’ serum 54 southeastern european medical journal, 2019; 3(1) discussion we recently showed that low dietary se content increases oxidative stress, which is possibly caused by decreased gene expression of the antioxidant enzyme gpx1 in thoracic aortic tissue without changes of the same enzyme in serum samples (13). gene expression of other antioxidative enzymes (cu/zn sod and catalase) did not change significantly. gpx3 is a better isoform of gpx for determination of the antioxidant effect of se in blood samples, so we decided to measure the more appropriate gpx isoform, which is readily detectable in the blood (25). the main findings of this study are the following: a) serum concentration of the antioxidant enzyme gpx3 depends on the concentration of se in food; b) our study once again confirmed that food with sufficient levels of se (according to the world health organization (who) and the american institute of nutrition (ain)) is richest with the antioxidant gpx3, which has a protective role in the body; c) different concentrations of se have no effect on body weight. trace elements are essential for living organisms because they are necessary for normal function of the organism, its growth and performance of its metabolic functions. amounts in the human body range from 1.5 mg to 4.5 g. it is characteristic for trace elements that very small amounts of a trace element affect the condition of the entire organism (26, 27). levels of se in food can vary in different parts of the world and countries (28, 29). intakes of se are high in america and japan, and much lower in europe, particularly eastern europe (30). it was recently shown that se has an important role in antioxidant selenoproteins used for protection against oxidative stress initiated by ros and its compounds in different types of cancers, cardiovascular diseases, immune system diseases and aging (31, 32). the world health organization published findings that a diet which contains 0.1 mg se per kilogram of food is sufficient for normal function of the organism (33), while the american institute of nutrition (ain) and the testdiet® ain-93 growth purified diet indicate that 0.24 mg se/kg in food is optimal (21). according to these guidelines, we determined the values of low (0.030 mg/kg se) and normal (0.363 mg/kg se) levels of se for our research. measuring se levels in blood samples seems to be a good indicator because plasma and serum contain about 75% of the se. the level of se found in these samples is directly related to recent dietary intakes (29). our research has shown that consuming food containing sufficient amounts of se according to the guidelines provides a significantly higher level of gpx3 antioxidants in plasma compared to foods with low se concentration (figure 2). such findings indicate that se is directly responsible for increasing the antioxidative status of gpx and for lowering oxidative stress. se and its protective role against disease are mostly related to the removal of free radicals and the enzymatic breakdown of oxygen metabolites (34). for example, in tuberculosis patients, reduced oxidative stress was caused by ros generation with se supplementation (35). of all types of tissue, the thyroid gland has the highest se concentration, and in the form of gpx3, se protects thyroid cells from free radicals and oxidative stress (36, 37). it was shown that in hashimoto's thyroiditis se supplementation significantly lowered thyroid peroxidase autoantibody titer after 3 months (38). furthermore, studies showed that se has a beneficial effect on lowering the risk for different types of cancer (3943). it can reduce oxidative damage and prevent dna damage (44). it can be used as alternative medicine by cancer patients undergoing radiotherapy (45 47). in addition, cellular and molecular processes that might be involved in the anti-cancer effects of se are stabilization of the immune response, induction of programmed cell death and inhibition of angiogenesis (44). lack of the se causes irreversible brain injury (48). similar, lower serum se was also found in children with epileptic seizures or febrile seizures and in adults with epileptic seizures (49, 50). seemedj 2019, vol 3, no. 1 low selenium diet in male sprague-dawley rats’ serum 55 southeastern european medical journal, 2019; 3(1) se has also been proved to be important for maintaining the immune system. it has been shown that se in a concentration of 400 μg per day significantly increased the number of t-cells (51), and supplementation with 100 μg per day for 6 months significantly increased the response to antigen challenge (52). se deficiency may cause a slower immune response because of the negative influence it has on immune cells (53). ros is involved in regulating the synthesis of adhesion molecules on endothelial cells, and is essential for inflammatory responses during the early stages of a disease (54, 55). the expression of intercellular and vascular adhesion molecules increases significantly during inflammation and is involved in the firm adhesion of leukocytes and endothelial cells (54). se nutritional status can directly influence vascular endothelial cell functions, e.g., under low se conditions, aortic endothelial cells exhibit increased platelet activating factor biosynthesis, which causes vascular disorders that are affected by increased oxidative stress (56). it was also found that the production of prostaglandins was significantly decreased in se-deficient endothelial cells, which are associated with the pathophysiology of several inflammatory diseases through significantly increased biosynthesis of thromboxane b2 (txb2) and 15hydroperoxyeicosatetraenoic acid (15-hpete) (57, 58). crucial role of se in oxidative stress and endothelial influence has been demonstrated by the study stupin et al. (2017), which showed that increased oxidative stress affects a nomediated response in low-se aortas probably due to decreased no bioavailability (13). selenoproteins regulate vascular tone by establishing a balance between superoxide anion and nitrogen oxide (7), which was found to be the case in this study as well. numerous studies have shown the important role that se plays in different health/disease states. further research is necessary to make use of the benefits of this important element as much as possible. observing the specific role of se in the endothelium, glutathione peroxidase was found to have an indispensable role in the maintenance of normal vascular function. specifically, low se decreased tissue expression of gpx1 and blood concentration of gpx3, which caused an increase in oxidative stress and disrupted endothelial function (figure 3). figure 3. negative effect of low se levels on changed vascular antioxidant status and consequent endothelial dysfunction seemedj 2019, vol 3, no. 1 low selenium diet in male sprague-dawley rats’ serum 56 southeastern european medical journal, 2019; 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1;28(3):381-9. 58. weaver ja, maddox jf, cao yz, mullarky ik, sordillo lm. increased 15-hpete production decreases prostacyclin synthase activity during oxidant stress in aortic endothelial cells. free radic biol med. 2001; 1;30(3):299-308. seemedj 2017, vol 1, no. 2 coital urinary incontinence and female sexual function 11 southeastern european medical journal, 2017; 1(2) coital urinary incontinence and female sexual function 1 ivan radoja1, oliver pavlović1, nikica perić1, dunja degmečić2 1 department of urology, university hospital center osijek, osijek, croatia 2 faculty of medicine, josip juraj strossmayer university of osijek, university department of psychiatry, university hospital center osijek, osijek, croatia corresponding author: dunja degmečić, md, phd – dunja.degmecic@mefos.hr introduction the international continence society (ics) terminology for lower urinary tract dysfunction (lutd) from 2003, which can be divided into storage symptoms and voiding symptoms, defines urinary incontinence (ui) as a storage symptom and as the complaint of any involuntary loss of urine (1). ui can also be defined as the inability to control urination which is manifested in the range from temporary leakage of urine to complete involuntary voiding which can be seen as a social and hygienic problem (2, 3). the anatomical structures of the female reproductive and urinary system are closely related, which leads to the conclusion received: june 30, 2017; revised version accepted: october 10, 2017; published: november 24. 2017 keywords: urinary incontinence, coitus, sexual dysfunction, sexuality that urinary problems interfere with sexual health in females (4). according to the current world health organization definition, sexual health is a state of physical, emotional, mental and social well-being in relation to sexuality, it is not merely the absence of disease, dysfunction or infirmity (5). the normal male sexual response cycle can be divided into libido, erection, ejaculation, orgasm and detumescence (6). sexual response cycle in women can be divided into libido, arousal, orgasm and satisfaction (7). damage of the integrity of the sexual response cycle, which is essential to human sexual functioning, may result in sexual dysfunction (8). female sexual dysfunction has been characterized as a persistent, recurrent problem abstract urinary incontinence (ui) is an everyday problem among a large proportion of adult women. the prevalence of ui ranges between 15% and 25% and the rate of incidence for each type of ui ranges between 10% and 58%. because women are concerned about ui during sexual intercourse, it has a negative effect on female sexual health. the incidence of ui during sexual intercourse in incontinent women has been reported to range between 10% and 27%. the prevalence of female sexual dysfunction is estimated to be 43%. there are conservative and surgical methods of treating ui but the number of published scientific articles dealing with the assessment of the effects of these types of therapy on improving sexual health in women treated for ui is deficient. (radoja i, pavlović o, perić n, degmečić d. coital urinary incontinence and female sexual function. seemedj 2017; 1(2); 11-18) seemedj 2017, vol 1, no. 2 coital urinary incontinence and female sexual function 12 southeastern european medical journal, 2017; 1(2) with sexual response, sexual desire, arousal, orgasm or dyspareunia and vaginismus that distresses the affected or strains the relationship with their partner (9). as reported by the international urogynecological association and the international continence society in 2010, incontinence during sexual intercourse or coital urinary incontinence (cui) is defined as the complaint of involuntary loss of urine during coitus (10). the objective of this study was to review the available evidence on incidence, prevalence, pathophysiology and treatment of ui and coital urinary incontinence (cui) with the attempt to determine the impact of ui and its physical and psychological consequences on women's sexual health and function in order to advise in a timely manner, prevent and treat disorders associated with bladder control issues. urinary incontinence – definition, etiopathogenesis, epidemiology women seldom visit general practitioners regarding incontinence problems which occur during or independently of the sex act, being that they are not familiar with the possibilities of treatment and mainly because a lot of women are ashamed to admit their “condition” unless they are precisely inquired by specialists or asked to “fill out” related questionnaires (11, 12). ui has a negative impact on sexual health irrespective of whether or not unwanted urine leakage occurs during sexual intercourse (4). ui products (e.g. incontinence pads, diapers) keep women dry but wearing them all day can cause chronic skin changes of the genital region which leads to discomfort and pain especially throughout sexual intercourse (13). furthermore, worrying about urinary leakage and odor during intercourse can cause feelings of anger, sadness, embarrassment, despair and low selfesteem. consequently, women abstain from having intercourse, which can cause reduction in the frequency of sexual relations, reduction of sexual desire and ability to achieve an orgasm. what's more, men no longer find their partner sexually attractive and some men even experience erectile dysfunction (14, 15). the pathophysiology and frequency of cui and impact of cui on quality of life are still not conclusive because there is limited research data. ui is not a disease but a symptom that is caused by disorders of pelvic floor muscles, the urethral sphincter and bladder. ui is most commonly divided into 3 types: stress urinary incontinence (sui) which is most prevalent (51%), urge urinary incontinence (uui) (10%) and mixed urinary incontinence (mui) (39%) (16). in women with sui, involuntary urine leakage occurs during coughing, laughing, sneezing and other physical activities (e.g. exercise, lifting heavy objects, sexual intercourse). uui is associated with an overwhelming urge to urinate that cannot be suppressed or delayed and is caused by detrusor overactivity. mui is defined as the involuntary leakage of urine accompanied by a sense of urgency during physical exertion, exercise, sneezing or coughing. urine loss during sexual activity may occur during arousal, penetration, orgasm, or resolution. current research suggests that in women with sui episodes of urine leakage are more likely to occur with penetration due to pressure on the bladder but in the case of uui urine is more likely to leak during orgasm (17). in this fashion, we can determine two types of cui: incontinence during penetration and incontinence at orgasm (17). risk factors for the occurrence of ui are sex, age, cigarette smoking and high body mass index (bmi) (18). ui is three times more common in women than in men regardless of the type (19). the prevalence of ui increases with age and in women between 15 and 60, it ranges from 10 to 25% (20). in older age groups, the number of incontinent women is higher although the vast majority of older women have acceptable control over their urination. among women aged 60 and over, prevalence of ui is 38% (21). the prevalence of female sexual dysfunction is estimated to be 43% and it increases with age (22). the incidence for each type of ui in females ranges between 10% and 58% depending on the observed population. it is generally accepted that the etiology of ui in women is associated with vaginal delivery, especially the first vaginal birth, menopause and surgical procedures in the pelvis and abdomen (23). the particular obstetric event that causes incontinence has not been found but it is most likely associated with newborns with an excessive birth weight and seemedj 2017, vol 1, no. 2 coital urinary incontinence and female sexual function 13 southeastern european medical journal, 2017; 1(2) difficult deliveries marked by prolonged pushing phases with or without instrumentation, which lead to nerve and muscle damage that provide a physiologic basis for this association (24). it is important to note that not all women who have had vaginal delivery experience symptoms of ui but they have more bothersome symptoms than women who have never had children. hormone changes in menopause and surgical procedures can also affect muscle strength in the pelvic region. cigarette smoking can induce chronic obstructive pulmonary disease with chronic cough that in time weakens the lower pelvic floor muscles and causes symptoms of sui but on top of that, ingredients of cigarette smoke can cause urinary bladder mucous membrane irritation leading to urgency and uui (25, 26). high bmi is a strong independent risk factor for ui and the mechanism of the onset of the disorder is that excessive body weight leads to an increase in intra-abdominal pressure which causes increased intravesical pressure, expanded mobility of the bladder neck and urethra, and also causes instability of the bladder detrusor, all leading to involuntary urine leakage (27). menopause and partner status are important predictors for female sexual dysfunction (28). interrelation of urinary incontinence, emotional, mental and sexual health ui has a negative impact on the quality of life of women, emotional health, physical and mental condition, impairs relationships, affects careers, and is also an additional financial burden (29, 30, 31). all incontinence types are associated with low self-esteem and a higher probability of psychiatric disease (32). the studies show higher levels of anxiety and psychological stress in women with ui (33). nowadays women lead active lives and are trying to maintain a healthy body condition and normal sex life regardless of age. ui has been recognized as one of the predictors of female sexual dysfunction considering excessive acetylcholine release on the bladder during intercourse, increased intraabdominal pressure with alteration of the urethrovesical angle and elevation of the bladder neck during intercourse, because of the fact that squamous epithelium of the trigone and urethra becomes thin and blood flow decreases with reducing estrogen in older postmenopausal women and the function of the main pelvic floor muscles deteriorates with age (34, 35). urine leakage during orgasm may be caused by involuntary detrusor contraction and relaxation of the urethra (36). involuntary urine leakage during sex is mainly a female problem because when a man has an erection the internal sphincter at the base of his bladder closes so urine can’t pass into urethra (37, 38). between 25 and 50% of women with ui experience decreased libido and decreased frequency of sexual activity because of the shame and fear of incontinence (39, 40). the incidence of ui during sexual intercourse in incontinent women has been reported to range between 10% and 27% (41). recent studies reported high prevalence of cui with results between 60% and 67% (42). the pelvic organ prolapse/urinary incontinence sexual questionnaire (pisq) and its short form version pisq-12 are validated condition-specific female sexual function questionnaires purposively developed to assess sexual function in women with ui and/or pelvic organ prolapse (43, 44). patients with a mui diagnosis had significantly lower mean pisq-12 scores than the ones with sui and uui, and patients with sui had significantly lower pisq-12 scores than those with uui (45). on the other hand, one study showed that among sexually active women with ui, sexual function as assessed by the pisq-12 does not differ according to type of incontinence (46). the result of one study was similar to most other research and actually established that the rate of overall female sexual dysfunction in women with uui was higher than in those with sui, that cui was more frequent in women with uui than in those with sui, but that feelings of pain and impressions of more involuntary urine leakage during sexual intercourse were more common in women with sui than in those with uui (40). some researchers have found that cui was present to a greater degree in young women than in elderly women and more frequent in women younger than 60 years of age (40, 47). in one study, 80% of women had incontinence during penetration, 93% had incontinence at orgasm and 92% had seemedj 2017, vol 1, no. 2 coital urinary incontinence and female sexual function 14 southeastern european medical journal, 2017; 1(2) incontinence in both cases, indicating cui as a common symptom during sexual activity in women with sui and suggesting urethral dysfunction as the possible explanation of cui (11). maximal urethral closure pressure < 30 cmh2o was associated with cui pointing out that urethral function plays a vital role in maintaining continence during coitus (48). in another report, 46% of women had uui which led to the termination of sexual activity without orgasm and the rate of female sexual dysfunction was detected to be higher in women with uui compared to that in the general population (49). gynecologists and urologists need to pay more attention to sexual dysfunction in women with ui in their clinical practice. additionally, one important thing to take into account is detailed medical history and sexual history of the occurring disorders during intercourse. treatment options once inflammation or anatomical disorders of the urinary system, neurological disease or cancer are ruled out, the following conservative and surgical treatment methods are available, most of which are scientifically proven with a high level of evidence and explained in the 2017 european association of urology guidelines: open communication with one’s sexual partner, counseling with a therapist who specializes in sexual and relationship problems, bladder training, urinating prior to sexual intercourse, deferring intercourse, interrupting intercourse prematurely, avoiding certain positions, hurrying through sex, avoiding orgasm, regular physical activity and weight loss, smoking cessation, exercises to strengthen pelvic muscles, drug therapy (anticholinergics and ß3-agonist), periurethral bulking agents, intravesical onabotulinumtoxin a injections, midurethral synthetic sling insertion, colposuspension and pubovaginal slings (50, 51, 52, 53, 54). the success rate is around 51% to 91% depending on the method, definition of cure and follow-up of every patient (55). in most cases, women try to perform the exercises of the pelvic floor on their own, in the comfort of their home, but without much success because they're not doing the exercises correctly. the efficiency of exercises can be increased if women exercise together with a specialized pelvic floor therapist. oral anticholinergics and ß3-agonists are the mainstay of pharmacological treatment, but they have side effects leading to a high discontinuation rate. it has been observed that in 60% of cases anticholinergic therapy leads to improvement of disorders during intercourse in women with ui (56, 57). intradetrusor injection of 100 units of onabotulinumtoxin a significantly decreased the daily frequency of ui with clinically relevant improvement of symptoms and health related quality of life in patients inadequately treated with anticholinergics (58, 59). the midurethral synthetic sling insertion has become the “gold standard” of surgical treatment of sui (60). in women who do not wish to have surgery or in whom surgical options are restricted (e.g. after irradiation), bulking agents should be considered as an alternative strategy because they are a minimally invasive approach to treat sui (61). their use should not be proposed as first-line treatment in women seeking a permanent cure for both primary and recurrent sui (61). conclusion ui is associated with a profound sense of humiliation and it is subject to social stigma and prejudices (4). ui and incontinence during sexual intercourse is still a taboo subject and many women feel uncomfortable talking openly about it. according to some estimates only 20% of women with ui seek help because of the problems that arise during sexual intercourse. there are many methods women can use to reduce the possibility of uncontrolled leakage during sex while working on a durable solution. it isn't an easy topic to discuss with one’s sexual partner but the talk is necessary if the outcome is returning to the previous pleasurable sexual life. the treatment of ui and incontinence during sexual intercourse requires multidisciplinary teamwork and cooperation among specialists, sexual partners and society. conservative and surgical treatment of ui, irrespective of the type, can improve quality of life and enhance seemedj 2017, vol 1, no. 2 coital urinary incontinence and female sexual function 15 southeastern european medical journal, 2017; 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189:48-54. seemedj 2017, vol 1, no. 1 weight status and body composition in freshman students… 50 southeastern european medical journal, vol 1, 2017. weight status and body composition in freshman students at the college of applied sciences “lavoslav ruzicka” in vukovar, from 2008 to 2016 1 nebojša nešić1, erna davidović cvetko1, vesna šeper1 1 department for health studies, college of applied sciences lavoslav ruzicka in vukovar, vukovar, croatia corresponding author: erna davidović cvetko, msc, phd erna.davidovic@gmail.com received: march 24, 2017; revised version accepted: april 23, 2017; published: april 24. 2017 keywords: obesity, body fat percentage, waist to hip ratio, body mass index abstract aim: the aim of the study is to analyze the common occurrence of different weight categories among firstyear students at lavoslav ruzicka college for applied sciences in the city of vukovar, as well as to make an assessment of their body composition. methods: during the period from 2008 to 2016 there were 710 first-year students (461 women and 249 men) whose height and weight were measured, and body composition assessed, by bio-electric impedance analysis. results: most of the subjects were of normal weight (74.5%), while the ratio between the weight status categories of men has proven to be significantly different from that of women. it was established that 8.9% of women as opposed to 2% of men were below normal weight, while there were more obese individuals among men (25.7%) than there were among women (15.4%). however, the percentage of obesity was similar in both sexes, being 5.4% in women and 5.2% in men. there was no significant difference in the ratio between the weight categories during the measurement period. conclusion: most of the students at the college for applied sciences fall in the normal weight category. there are more obese individuals among men, while among women there are more of those with lower average body weight. the dominance of obesity and the overall ratio between the weight categories and the body composition in the category of first-year students have not changed significantly during the period between 2008 and 2016. (nešić n, davidović cvetko e, šeper v. weight status and body composition in freshman students at the college of applied sciences “lavoslav ruzicka” in vukovar, from 2008 to 2016. seemedj 2017;1(1);50-58) seemedj 2017, vol 1, no. 1 weight status and body composition in freshman students… 51 southeastern european medical journal, vol 1, 2017. introduction morphological characteristics are often the subject of research due to the great impact they have on health and disease development. obesity and fat accumulation are intertwined with hypertension, hyperlipidemia and hypercholesterolemia, as well as low levels of high-density cholesterol and hyperglycemia – all of which, among many other factors, are associated with cardiovascular problems (1). of all the previously mentioned conditions, obesity has by far the most important influence on the quality of life (2,3). in the usa, over one third of adults and around 17% of children are obese (4), while studies on european nations showed that around 17% of adults are obese, with some variations among countries (5). as for studies on students and younger adults, some data show that there are around 22% overweight students (6), while 50% of people aged 18-24 are subject to at least one risk factor for the development of cardiovascular diseases (7). it has been proven that being overweight and obese during pregnancy are conditions which increase the risk for the child itself to become overweight or to develop obesity. on the other hand, girls who are overweight during their adolescence are more prone to gaining weight in pregnancy, demonstrating a vicious cycle (8). the consequences of overweight and obesity not only affect overweight and obese persons, but society in global terms. the socioeconomic results of obesity include higher expenses for treating the diseases resulting from being obese (9-11). finally, obesity greatly increases the risk of mortality (12,13). therefore, preventive actions are very important, including education about diet, physical exercise and a healthy way of life. anthropometrical research conducted during the croatian adult health survey in 2003 (14) show that 58.5% of the adult population (over the age of 18) is overweight, whereas 20.4% is obese. because of such widespread adult obesity, there are some predictions that by the year 2030 37% of men and 48% of women will be obese—if the same trends towards increased obesity continue (14). the aim of this research is to analyze the common occurrence of different weight categories among first-year students at the lavoslav ruzicka college of applied sciences in the city of vukovar, and to assess their body constitution. additional goals of the study are the following: (1) the analysis of the weight status and the body constitution of students who began their studies within this institution between 2008 and 2016 in order to determine if there have been some significant changes; and (2) the examination of occurring variations in the weight status and body composition between the sexes. methods and materials study design during the period from 2008 to 2016, 710 first year students (461 women and 249 men) volunteered to participate in the study. every participant was measured once, during their first academic year. the average age at the time of the measurement was 19 (ranging from 18 to 26). measurements the subjects’ height was measured using standard techniques and equipment. weight and body composition were measured and assessed by bio-electric impedance analysis using the body composition analyzer gaia 359 (jawon medical, korea). this was performed in the morning hours after overnight fasting by a trained technician, following the device manufacturer’s directions. measurement results included weight, body fat mass (bfm), body fat percentage (bf%) and soft lean mass (slm). the body mass index (bmi) was calculated by the following equation: weight (in kilograms) divided by height (in square meters). whr (waist to hip ratio) was calculated by the following equation: waist circumference divided by hip circumference. seemedj 2017, vol 1, no. 1 weight status and body composition in freshman students… 52 southeastern european medical journal, vol 1, 2017. weight status was defined following current recommendations from the who, according to which a bmi between 18.5 and 24.9 represents normal weight, lower values of bmi are considered being underweight, while a bmi over 25 is considered being overweight and a bmi of 30 or higher is considered obesity (15). statistical analysis statistical analysis was carried out using ibm spss statistics 22. statistical significance was set at p<0.05. since all variables failed in the kolmogorov-smirnov test of normal distribution, they are presented as median and 95% confidence interval (ci) for median. the ratio between the weight status categories between sexes and during the year of measurement was tested by the chi-square test. the mannwhitney u test was used for testing the differences in the values of measured variables between sexes. spearman’s coefficient of correlation was used to calculate the correlation between bf% and bmi, and between bf% and whr. results bmi and body composition, as well as whr, for the total sample and by sex, are provided in table 1. the difference between the sexes was statistically significant in all variables (table 1). men had a higher value of bmi compared to women, but the range of results for bmi in women was wider than in men, so that the highest bmi measured was as follows: for women, it was 44.1 kg/m2, and for men it was 36.3 kg/m2; the lowest measured bmi was similar for both sexes (table 1). whr was also higher in men than in women, but bf% showed that women have more fat content compared to men. the range of bf% showed that fat content in both sexes ranges from very low (4.3%) to over 37% for men and over 45% for women. there was also a significant difference between sexes in the distribution along the weight status categories (p<0.0001). the difference between sexes in distribution along the weight status categories are shown in figure 1. the highest predominance was in the category of normal weight for both sexes, with around three quarters of participants (75.7% of women and 72.3% of men). in the underweight category, the women prevailed with 8.9%, whereas only 2%of men were in this category. a higher predominance of men compared to women was registered in the category of a bmi value over 25 (overweight and obese), with 25% of men vs. 15% of women. since similar proportions of men and women were obese (5.4% of women and 5.2% of men), the difference in the proportion of subjects with a bmi higher than 25 is consequent to the predominance of overweight subjects rather than obese ones. distribution along the weight status categories through the years is presented in figure 2. a chisquare test did not show any significant difference between the different measurement years (p=0.9). distribution through the weight status categories was similar during the entire period of the study, with some small changes, mostly regarding the prevalence of terminal categories: category obese, and category underweight. secular trends for men and women in bmi, whr and bf% are shown in figures 3-5. while there were no significant differences during the years of measurements for any of the variables, distinctive differences between the sexes remained present throughout all the measurement years. spearman’s correlation yielded a positive correlation of 0.532 (p<0.001) between bf% and bmi, and a positive correlation of 0.582 between bf% and whr. when calculated separately by sex, the correlation between bf% and bmi for men was 0,803 (p<0.001), and for women 0.924 (p<0.001). the correlation between bf% and whr for men was 0,983 (p<0.001) and for women 0.996 (p<0.001). discussion the main finding of the present study is that the distribution across the weight status categories is significantly different between sexes (table 2). although there was a similar proportion for the subjects of both sexes of normal weight, there seemedj 2017, vol 1, no. 1 weight status and body composition in freshman students… 53 southeastern european medical journal, vol 1, 2017. are more overweight individuals among men, while among women there are more of those with lower average body weight. it has also been found that the prevalence of obesity among those overweighted is similar in both sexes (table 2). the result of the similar prevalence of normal weighted among both sexes presented here complies with the results of other similar studies made throughout the student populations in india, saudi arabia and the usa, with the exception of the published results in listed countries showing lower portions of subjects of normal weight in their samples (1618). the portion of subjects with normal weight was around 20% lower than in the present study. a higher prevalence of underweight women as opposed to men was also reported in the study made by zhang et al. (19) and sira and pavlak (18). these results are similar to the results of studies performed at some other high schools or universities (16, 17,20). kritsotakis et al. (21) also reported lower values of bmi in women than in men. in the paper published by kantanista et al. (22), the authors concluded that under weighted adolescent girls are more satisfied with their body than those who are of normal weight and/or overweight. yatsuya et al. (23) reported a higher prevalence of obesity in men than in women in europe, which complies with the results of our study. in their study, the prevalence of obesity was assessed in different world regions, and the second highest rate was noted in europe. the results of research done in india (16), saudi arabia (17), usa (18) and brazil (20) show the prevalence of obese individuals among students of different races and ethnicities of about 6-15%, while the study of prevalence of obesity in 22 developing countries reached results similar to our study, which are 56% (24). great differences in the prevalence of obesity throughout europe were reported in comparisons made by borsika et al.(25). they reported that the highest obesity prevalence is present in hungary (19.5%), the russian federation (15.2%) and romania (14.7%), while the lowest obesity rates are in norway (6.2%), france (7%) and italy (7%). similar results were reported in the study by van viet ostapchouk et al. (5). from the study by fišter et al. (26), it could be concluded that the number of obese individuals rises later in life, while according to musić milanović et al. (27) the critical years when people are starting to become obese are up to 35, when most changes from overweight to obesity are indeed recorded. huang et al. (28) reported obesity prevalence rates at the university of kansas to be similar to those in our results. however, some recent studies reported lower proportions of obesity among university students in croatia of up to 3% (29-31). similar reports of overweight/obesity predominance from neighboring countries (32-34) showed that there are great variations between and within the countries of our region: at the university of novi sad overweight rates reported are 33.5% in men and 7.5% in women (32), while at the university of niš overweight prevalence is 38.18% in men and 7.95% in women (33). rašeta et al. reported 22.4% of overweight students at the university of banja luka (34). it would be interesting, in our opinion, to compare the weight status and body composition of students of medical science and kinesiology as opposed to those who study social or natural sciences. since medical students and students of kinesiology should be more aware of the risks and potential dangers of unhealthy weight, it would be interesting to see if their weight status is different in comparison to others. something similar was done by rašeta et al. at the university of banja luka (34), where obesity rates among students of three different faculties were compared: the faculty of physical education and sport, the faculty of economics and the faculty of medicine. results showed that the students of the faculty of economics had the highest value of bmi and bf%, while the medical students had the highest whr. other very important values for weight status assessment are whr and bf%, which are considered more accurate measurements of body weight type and body composition than bmi, because bmi cannot distinguish between weight associated with fat and weight associated with fat-free mass (35). most of the subjects in the present study had values of whr and bf% within the recommended ranges for health maintenance (figures 4 and 5). since those measures are predictive factors for seemedj 2017, vol 1, no. 1 weight status and body composition in freshman students… 54 southeastern european medical journal, vol 1, 2017. metabolic syndrome and a series of table 1. descriptive statistics. total (n=710) women (n=461) men (n=249) median (95% ci) min-max median (95% ci) min-max median (95% ci) min-max p* bmi (kg/m2) 22.10 (21.9-22.5) 15.9-44.1 21.5 (21.1-21.8) 15.9-44.1 23.4 (22.9-23.9) 16.3-36.3 <0.0001 whr 0.75 (0.74-0.76) 0.64-0.98 0.73 (0.72-0.74) 0.66-0.95 0.78 (0.78-0.80) 0.64-0.98 <0.0001 bf% 22.7 (22.2-23.5) 4.3-45.4 25.1 (24.5-25.7) 4.3-45.4 17.4 (16.5-18.4) 4.3-37.0 <0.0001 p* p value obtained by performing mann-whitney test for difference between sexes bmi body mass index; whr waist to hip ratio; bf% body fat percentage figure 1. difference between sexes in distribution along the weight status categories. absolute frequencies in each category are presented below the graph. chi-square test showed significant difference between men and women (p<0.0001) figure 3. secular trend for men and women in bmi. figure represents differences between sexes in bmi from 2008 to 2016 expressed in median (with 95% ci for median) values. figure 2. distribution along the weight status categories through the years. absolute frequencies in categories are presented below the graph. chi-square test did not show any significant difference between the different measurement years (p=0.9) 45 40 35 30 25 20 15 10 5 0 year b m i (k g /m 2 ) 2008 2009 2010 2011 2012 2013 2014 2015 2016 sex female male * seemedj 2017, vol 1, no. 1 weight status and body composition in freshman students… 55 southeastern european medical journal, vol 1, 2017. cardiovascular problems (35), it is very important that the values were within healthy limits. some researches show that, similar to bmi, values for pbf and whr rise as people age (36). the body composition (presented in table 1) of the subjects shows that they have appropriate mass of fat and muscles, and their body composition is in accordance to weight status based on bmi value. in other words, the prevalence of obesity based on bmi distribution would probably be similar if they were distributed according to the bf% or slm. results reported by grygiel-gorniak et al. (37) show similar values of bmi, whr and bf% with the results of the present study, as well as the similar differences between sexes. our findings regarding differences in bmi and bf% between men and women showed that men had higher bmi, but lower bf% compared to women. this could be ascribed to the greater muscle mass in men. however, the correlation between bf% and bmi showed a statistically significant high positive value, indicating a strong connection between those two variables. correlation was slightly lower in men than in women. there was also high correlation between bf% and whr in both sexes. collins et al., in their study of association of bmi and bf% among bmi-defined non-obese middle-aged individuals, found that the bmi category was not concordant with the %bf classification for 30% of the population. the greatest discordance between %bf and bmi was observed among %bf-defined overweight/obese women (38). a strong correlation of bmi and bf% in young women was reported in the study of bakir et al. (39). they obtained correlation coefficients between bmi and bf% of 0.74 for women aged 18-30 years. proportions of weight categories were not significantly different over the years in which measurements were made. students that choose to attend the college of applied sciences in vukovar are similar in weight status throughout the years. this result is different compared to predictions of increase in obesity prevalence, and shows a steady state in the weight status of first-year students during the years examined, without any increase of obesity. it is possible, however, that it might have been too short a period for potential trends to reveal themselves. based on the presented results, the conclusion could be made that most of the freshman students at the college of applied sciences fall in the category of normal weight, with an overweight prevalence of around 19-20%, including around 5% obese persons among them. there are also 6.5% of those who are underweight. there is a higher tendency toward the prevalence of overweight persons among men, while among women there is a higher tendency for underweight prevalence. the prevalence of obesity and the overall distribution across the weight categories, as well as the body composition of the first-year students have not changed during the period from 2008 to 2016. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. grundy sm, brewer hb, cleeman ji, smith sc, lenfant c. definition of metabolic syndrome. circulation 2004;109(3):433-438. 2. ford es, li c. 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wang c, chiang c, zhang y, uemura m, osako a, ozaki y, aoyama a. global trend in overweight and obesity and its association with cardiovascular disease incidence. circ j. 2014;78(12):2807-2818. 24. peltzer k, pengpid s, samuels t, özcan nk, mantilla c, rahamefy oh, gasparishvili a. prevalence of overweight/obesity and its associated factors among university students from 22 countries. int j environ res public health. 2014;11(7):7425-7441. 25. rabin ba, boehmer tk, brownson rc. cross-national comparison of environmental and policy correlates of obesity in europe. eur j public health. 2006;17(1):53-61. 26. šatalić z, colić barić i, keser i. diet quality in croatian university students: energy, macronutrient and micronutrient intakes according to gender. int j food sci nutr 2007;58(5): 398-410. 27. rakovac m, pedisic z, pranic s, greblo z, hodak d. sociodemographic and lifestyle correlates of health-related quality of life in croatian university students. arqol 2013;8(4):493-509. 28. huang ttk, harris k j, lee re, nazir n, born w, kaur h. assessing overweight, obesity, diet, and physical activity in college students. j am coll health 2003;52(2):83-86. 29. pedišić ž, rakovac m, bennie j, jurakić d, bauman ae. levels and correlates of domain-specific physical activity in university students: cross-sectional findings from croatia. kinesiology 2014;46(1):12-22. 30. fišter k, kolčić i, musić milanović s, kern j. the prevalence of overweight, obesity and central obesity in six regions of croatia: results from the croatian adult health survey. collegium antropol 2009;33(1):2529. 31. musić milanović s, ivičević uhernik a, fišter k, mihel s, kovač a, ivanković d. five-year cumulative incidence of obesity in adults in croatia: the crohort study. collegium antropol 2012;36(1):71-76. 32. simić s, vasić g, jakonić d. telesna visina, telesna masa i uhranjenost studenata univerziteta u novom sadu [body height, body weight and nutritional status in students of the university of novi sad]. med danas 2010;9(4-6):141–146. 33. stojanović d, višnjić a, mitrović v, stojanović m. risk factors for the occurrence of cardiovascular system diseases in students. vojnosanit pregl 2009;66(6):453–458. 34. rašeta n, đurić s, zeljković n, simović s, vujnić m. interrelations between body mass index, percentage of bodyfat, and waist-tohip ratio among different groups of students at the university of banja luka. fu phys ed sport 2016;14(3):331–345. 35. schneider hj, friedrich n, klotsche j, pieper l, nauck m, john u, dorr m, felix s, lehnert h, pittrow d, silber s, volzke h, stalla gk, wallaschofski h, wittchen hu. the predictive value of different measures of obesity for incident cardiovascular events and mortality. j clinc endocrinol metab 2010;95(4):1777–1785. 36. tucak-zorić s, bilić čurčić i, mihalj h, dumančić i, zelić ž, majetić cetina n, seemedj 2017, vol 1, no. 1 weight status and body composition in freshman students… 58 southeastern european medical journal, vol 1, 2017. szirovicza l. prevalence of metabolic syndrome in the interior of croatia: the baranja region. collegium antropol 2008;32(3):659-665. 37. grygiel-górniak b, tomczak a, krulikowska n, przysławski j, seraszek-jaros a, kaczmarek e. physical activity, nutritional status, and dietary habits of students of a medical university. sport sci health 2016;2:261-267. 38. collins kh, sharif, b, sanmartin c, reimer ra, herzog, w association of body mass index (bmi) and percent body fat among bmi-defined non-obese middle-aged individuals: insights from a populationbased canadian sample. can j public health 2016;107(6) :e520-e525. 39. bakir m, al-bachir m, hammad kb, habil km, ahmad h. the accuracy of currently used who´s body mass index cut-off points to measure overweight and obesity in syrian women: a correlation study. ebph 2016;13(3):e11852-1 e11852-9. seemedj 2019, vol 3, no. 2 clinical supervision in nursing in journals 45 southeastern european medical journal, 2019; 3(2) original article representation of topics regarding clinical supervision in nursing in national and international journals 1 ana budrovac 1, nada prlić 2 1 health centre osijek, croatia 2 faculty of dental medicine and health osijek, croatia corresponding author: ana budrovac, anabud94@gmail.com received: may 29, 2019; revised version accepted: dec 4, 2019; published: dec 16, 2019 keywords: clinical supervision, database, nursing, supervisory abstract aim: the aim of this paper was to examine the representation of topics of clinical supervision in nursing in national and international journals. methods: a databases medline, ebsco and hrčak were retrospectively researched. the research encompassed nursing journals published from 2011 to 2015. mesh keywords were used as well. the availability of articles in full text was examined by pubmed central and by using pero, a search engine for electronic resources online, which is an online source of journals with articles available in full text to the croatian scientific and academic community. a total of 168 articles and 54 journals were included in this research. results: the topic of clinical supervision in nursing has not been found in any of the papers published in croatian journals. there were 168 scientific papers about clinical supervision in nursing in 54 international journals. in the “journal of nursing management” there was a trend of a decreasing number of published scientific papers (cochran–armitage test p< 0.0001). in the medline database there were 43 journals. most journals were published in the usa, 23 of them. a total of 29 articles are available in full form. ovidsp enables access to the highest number of articles, 18 (62.1%), while pero search engine found no articles in full form. conclusion: in national journals in croatia there are no topics regarding clinical supervision in nursing, but there are some in international journals. (budrovac a, prlić n. representation of topics regarding clinical supervision in nursing in national and international journals. seemedj 2019; 3(2); 45-55) seemedj 2019, vol 3, no. 2 clinical supervision in nursing in journals 46 southeastern european medical journal, 2019; 3(2) introduction definition of the word “supervise” is to watch somebody or something to make sure that work, etc is being done properly and that people are behaving correctly (1). that is why nurses very often have a negative attitude towards clinical supervision (cs). smith highlighted that it is not about controlling somebody; instead, it is about empowering staff. sabrage said it has been described in literature as “time for me” (2). in addition to that, fowler stated that nurses should sometimes put themselves before others (3), which gives cs a completely different meaning. there have been many attempts to define cs. according to the care quality commission, “the purpose of cs is to provide a safe and confidential environment for staff to reflect on and discuss their work, personal and professional response to their work. the focus is on supporting staff in their personal and professional development and in reflecting on their practice” (4). furthermore, the royal college of nursing states that the term “clinical supervision” is used to describe a formal process of professional support and learning which enables practitioners to develop knowledge and competence, assume responsibility for their own practice and enhance consumer protection and the safety of care in complex clinical situations. it is central to the process of learning and to the expansion of the scope of practice and should be seen as a means of encouraging selfassessment and analytic and reflective skills (5). bishop and sweeney described it as ”designated interaction between two or more practitioners within a safe and supportive environment, that enables a continuum of reflective critical analysis of care, to ensure quality patient services and the wellbeing of the practitioner” (6). despite these and numerous other definitions, many authors state that definition of clinical supervision is still unclear (7,8). butterworth et all justify this by explaining that there are many different definitions, especially among states because of socio-cultural impact (9). fowler explains that “the practice of clinical supervision will and should vary because the practice of nursing varies across different environments and patient groups” (10). except those, he describes some more factors, such as ward culture and organisation, ratio of the number of employees, employees’ needs and management (11). clinical supervision can be implemented in different ways. it can be implemented as a oneto-one relationship or group supervision. the other consideration is whether supervision is among peers, in a team or multidisciplinary. one-to-one is a fairly common model, where a more experienced nurse supervises a less experienced one. experienced specialist nurses can be involved in “peer supervision”, whereby staff of similar experience and profession “cosupervise” each other. however, a specialist nurse can also receive supervision from someone with greater experience, but who is not a nurse. there are three types of cs groups: peer, team and multidisciplinary groups. in peer cs groups, all staff members are of similar qualifications and experience (e.g. nurses, clinical managers, healthcare assistants, clinical nurse specialists). team supervision occurs within the established hierarchical clinical team. multidisciplinary supervision tends to occur where multidisciplinary staff work closely together (12). one of the three most frequently cited models was reported by winstanley and white – proctor's interactive model, highlighting the normative (managerial), formative (educative) and restorative (supportive) functions of supervision (13). the aim of the normative component is to support reflection on practise with an awareness of local policy and codes of conduct. formative component focuses on the development of skills, knowledge, attitudes and understanding. restorative component fosters resilience through nurturing supportive relationships that offer motivation and encouragement and that can also be drawn seemedj 2019, vol 3, no. 2 clinical supervision in nursing in journals 47 southeastern european medical journal, 2019; 3(2) upon in times of stress (14). that model has become the most used model (15). although the principles of clinical supervision are simple, its implementation is more difficult. it requires time, commitment, openness to selfreflection, admission of areas of weakness and a wiliness to develop and grow (3). many authors describe lack of time as the main barrier to clinical supervision implementation (16-20). fowler states that we must make time for clinical supervision, it needs to be viewed in the same way as mandatory training, days off or annual leave (21). there are many benefits of cs, some of them are: improved employee retention, better motivation and commitment to the organisation, maintenance of clinical skills and quality practice, improved communication among workers, increased job satisfaction, self-critique of clinical and cultural practice in a safe environment, development of strategies to address issues raised as part of critiquing and reflecting on practice, identification of strengths in practice, identification of learning opportunities to enhance further development of nursing practice, prevention of burnout, nursing leadership development (22). the aim of this paper was to examine the representation of clinical supervision topics in nursing in national and international journals. material and methods databases medline, ebsco and hrčak were retrospectively researched. the research encompassed nursing journals published from 2011 to 2015. mesh keywords “organization and administration” and “nursing, supervisory” were used for searching the medline database. subheading “standards” was used to restrict the heading “organization and administration” and searching was restricted to nursing journals. subheadings “standards”, “organization and administration” and “statistic & numerical data” were used to restrict the heading “nursing, supervisory” (figure 1). for searching the ebsco database, the title “clinical supervision” and subject term “nursing” were combined with boolean operator and (figure 1). keyword “supervizija”/ “supervision” was used for searching the database hrčak. results were restricted to the area of “biomedicine and healthcare” (figure 1). results in all databases were restricted to nursing journals (figure 1). nursing journals which did not have access to all or some parts of journal volumes were excluded from research. in all included journals, the main focus was on looking for all published articles and all articles about clinical supervision in the period from 2011 to 2015. the availability of articles in full text was examined by pubmed central and by using pero. a total of 168 articles and 54 journals were used in this research (figure 1). seemedj 2019, vol 3, no. 2 clinical supervision in nursing in journals 48 southeastern european medical journal, 2019; 3(2) figure 1. flow chart * use the keyword 'supervision' then continue to search heading organization & administration † global health action; global journal of health science; palliative medicine; studies in health technology and informatics; health service journal; advances in health care management; advances in neonatal care; giornale italiano di medicina del lavoro ed ergonomia; journal of the medical association of thailand; critical and resuscitation; journal of intellectual and developmental disability; international journal of evidence-based health care; journal of occupational rehabilitation ‡ journal of intellectual disabilities and offending behaviour; scandinavian journal of caring sciences; research on social work practice § nursing standard; australian nursing & midwifery journal; mental health nursing; british journal of nursing; american journal of nursing; insight; nursing times; nursing new zealand (wellington); tar heel nurse; rcm midwives ǁ jarna; nursing standard; mental health nursing; community practitioner; nosileftiki; british journal of nursing; communicating nursing research; practice nurse; australian nursing & midwifery journal ¶ miller a, buerhaus pi. the changing nature of icu charge nurses' decision making: from supervision of care delivery to unit resource management. joint commission journal on quality & patient safety. 2013;39:38-47. ** perspectives in psychiatric care; nursing & health sciences; journal of advanced nursing; nurse education today; nurse education in practice; journal of psychiatric & mental health nursing; journal of clinical nursing; journal of nursing management; issues in mental health nursing; australian critical care; international journal of mental health nursing †† gonge h, buus n. model for investigating the benefits of clinical supervision in psychiatric nursing: a survey study. international journal of mental health nursing. 2011;20:102-11. seemedj 2019, vol 3, no. 2 clinical supervision in nursing in journals 49 southeastern european medical journal, 2019; 3(2) cross wm, moore ag, sampson t, kitch c, ockerby c. implementing clinical supervision for icu outreach nurses: a case study of their journey. australian critical care. 2012;25:263-70. kenny a, allenby a. implementing clinical supervision for australian rural nurses. nurse education in practice. 2013;13:165-9. koivu a, hyrkas k, saarinen pi. who attends clinical supervision? the uptake of clinical supervision by hospital nurses. journal of nursing management. 2011;19:69-79. gonge h, buus n. is it possible to strengthen psychiatric nursing staff's clinical supervision? rtc of meta-supervision intervention. journal of advanced nursing. 2015;71:909-21. sharrock j, javen l, mcdonald s. clinical supervision for transition to advanced practice. perspectives in psychiatric care. 2013;49:118-25. carver n, clibbens n, ashmore r, sheldon j. mental health pre-registration nursing students' experiences of group clinical supervision: a uk longitudinal qualitative study. nurse education in practice. 2014;14:123-9. results the topic of clinical supervision in nursing has not been found in any of the papers published in croatian journals. there were 168 scientific papers about clinical supervision in nursing in international journals. the biggest number of journals about cs were published in 2011, a total of 46. the fewest were published in 2014, only 25. the “journal of nursing management” contained the most scientific papers about cs in 2011, specifically 19 of them. in that journal there was also a trend of reduced number of published articles in the period from 2011 to 2015 (cochran – armitage test p<0,0001) (table 1).. table 1. representation of topics regarding clinical supervision in nursing in international journals journal total number of published articles (proportion of articles dealing with clinical supervision) 2011 2012 2013 2014 2015 p* bmc nursing 24 (0) 28 (0) 29 (0) 50 (0) 85 (1) 0.34 nordic journal of nursing research & clinical studies /vard i norden 36 (0) 41 (0) 40 (2) 43 (0) 38 (0) 0.97 perspectives in psychiatric care 32 (0) 30 (0) 35 (1) 35 (0) 35 (0) 0.96 nursing & health sciences 76 (1) 71 (1) 75 (1) 73 (0) 71 (1) 0.74 international journal of nursing studies 166 (1) 163 (0) 168 (0) 170 (0) 198 (1) 0.93 journal of advanced nursing 253 (0) 256 (2) 266 (0) 267 (2) 253 (1) 0.53 nurse education today 182 (0) 191 (1) 282 (3) 268 (0) 270 (2) 0.6 nurse education in practice 105 (1) 114 (1) 170 (3) 120 (3) 93 (1) 0.56 journal of psychiatric & mental health nursing 136 (0) 111 (1) 122 (1) 113 (2) 96 (0) 0.58 journal of clinical nursing 395 (1) 385 (2) 358 (0) 368 (1) 368 (0) 0.32 connect: the world of critical care nursing 25 (0) 26 (0) 17 (0) 20 (1) 19 (0) 0.41 british journal of midwifery 149 (0) 169 (0) 150 (0) 168 (1) 162 (0) 0.49 journal of nursing education scholarship 28 (0) 26 (1) 36 (0) 18 (0) 20 (0) 0.54 contemporary nurse: a journal for the australian nursing profession 88 (0) 84 (0) 84 (1) 73 (0) 49 (0) 0.86 seemedj 2019, vol 3, no. 2 clinical supervision in nursing in journals 50 southeastern european medical journal, 2019; 3(2) table 1a. representation of topics regarding clinical supervision in nursing in international journals journal total number of published articles (proportion of articles dealing with clinical supervision) 2011 2012 2013 2014 2015 p* journal of nursing management 109 (19) 205 (3) 106 (0) 100 (0) 112 (4) <0.0001 australian critical care 102 (0) 80 (1) 79 (0) 97 (0) 90 (0) 0.49 international journal of mental health nursing 52 (2) 68 (0) 63 (3) 65 (0) 64 (0) 0.16 journal of nursing measurement 14 (0) 17 (0) 34 (0) 43 (1) 46 (0) 0.74 obzornik zdravstvene nege 37 (1) 28 (0) 30 (0) 26 (0) 26 (0) 0.98 klinisk sygepleje 29 (0) 31 (1) 34 (0) 28 (0) 29 (0) 0.48 assistenza infermieristica e ricerca:air 25 (0) 34 (2) 28 (0) 30 (0) 27 (1) 1.00 practising midwife 99 (1) 118 (3) 113 (3) 110 (7) 114 (6) 0.05 nursing economics 42 (0) 48 (0) 39 (0) 34 (0) 46 (1) 0.16 health care manager 43 (0) 37 (0) 41 (1) 38 (0) 47 (1) 0.35 jognn – journal of obstetric, gynecologic, & neonatal nursing 100 (0) 100 (0) 92 (2) 89 (0) 98 (1) 0.39 nursing management (harrow) 171 (1) 148 (3) 74 (2) 176 (1) 111 (1) 0.82 professioni infermieristiche 23 (0) 27 (0) 26 (0) 27 (0) 28 (1) 0.17 soins; la revue de reference infirmiere 255 (0) 185 (0) 172 (0) 156 (1) 140 (0) 0.37 cin: computers, informatics, nursing 132 (0) 106 (0) 104 (0) 97 (1) 99 (0) 0.43 women & birth: journal of the australian college of midwives 26 (0) 35 (0) 72 (0) 60 (1) 72 (0) 0.66 the gerontologist 73 (0) 91 (0) 107 (0) 115 (1) 121 (0) 0.58 worldviews on evidence-based nursing 26 (2) 25 (0) 19 (1) 48 (1) 46 (0) 0.11 queensland nurse 53 (0) 53 (0) 59 (1) 63 (0) 45 (0) 0.99 nursing administration quarterly 43 (3) 46 (0) 57 (2) 55 (0) 60 (1) 0.14 seemedj 2019, vol 3, no. 2 clinical supervision in nursing in journals 51 southeastern european medical journal, 2019; 3(2) table 1b. representation of topics regarding clinical supervision in nursing in international journals journal total number of published articles (proportion of articles dealing with clinical supervision) 2011 2012 2013 2014 2015 p* journal of nursing administration 106 (1) 112 (3) 122 (3) 116 (0) 121 (1) 0.38 nursing forum 32 (1) 33 (0) 34 (3) 32 (0) 35 (0) 0.45 canadian nurse 209 (0) 206 (1) 205 (2) 203 (0) 167 (0) 0.76 joint commission journal on quality & patient safety 69 (0) 65 (0) 70 (1) 71 (0) 72 (0) 0.98 journal of nursing care quality 45 (0) 51 (0) 45 (2) 48 (0) 53 (0) 0.96 midwifery 154 (0) 117 (0) 177 (1) 156 (0) 146 (0) 0.98 mcn, american journal of maternal child nursing 85 (0) 81 (1) 87 (0) 82 (0) 92 (0) 0.47 krankenpflege – soins infirmiers 158 (0) 163 (1) 147 (0) 171 (0) 202 (0) 0.44 online journal of issues in nursing 28 (1) 26 (1) 24 (0) 22 (0) 24 (0) 0.16 research in nursing & health 40 (0) 52 (1) 47 (0) 45 (0) 43 (0) 0.47 nursing leadership (toronto, ont) 33 (2) 45 (0) 44 (0) 30 (0) 18 (0) 0.05 issues in mental health nursing 127 (1) 133 (0) 142 (1) 128 (0) 138 (0) 0.30 public health nursing 64 (1) 63 (0) 62 (0) 62 (0) 75 (0) 0.15 journal of cardiovascular nursing 74 (1) 60 (0) 72 (0) 82 (0) 84 (0) 0.14 journal of emergency nursing 137 (1) 159 (0) 154 (0) 150 (0) 140 (0) 0.15 journal of the american medical directors association 107 (1) 183 (0) 200 (0) 150 (0) 213 (0) 0.10 aaohn journal 73 (1) 95 (0) 93 (0) 91 (0) 88 (0) 0.13 international nursing review 92 (1) 103 (0) 81 (0) 74 (0) 74 (0) 0.19 journal of the american association of nurse practitioners 82 (0) 86 (0) 89 (0) 83 (0) 112 (1) 0.19 mental health practice 220 (1) 206 (0) 185 (1) 177 (1) 186 (0) 0.78 total 5084 (46) 5216 (30) 5261 (41) 5216 (25) 5261 (26) 0.01 * cochran – armitage test seemedj 2019, vol 3, no. 2 clinical supervision in nursing in journals 52 southeastern european medical journal, 2019; 3(2) most of the journals in which scientific papers about cs were published were found in the medline database, specifically 43 of them. in the hrčak database there were no journals containing articles about cs in nursing (table 2). table 2. representation in databases medline, ebsco and hrčak of journals in which articles about clinical supervision in nursing were published database number (%) total number of journals journals in which articles about cs were published medline 1235 (100) 43 (3.5) ebsco 15337 (100) 22 (0.1) hrčak 437 (100) 0 in total, there were 54 journals in which articles about cs in nursing were published. most of the journals were published in usa, 23 (42,5 %) of them. slovenia, france, switzerland and denmark each had only one journal with articles about cs in nursing published (table 3). table 3. list of countries publishing journals in which articles about cs in nursing were published country publishing journal number (%) of journals in which articles about cs in nursing were published usa 23 (42.5) great britain 19 (35.1) australia 4 (7.4) canada 2 (3.7) italia 2 (3.7) slovenia 1 (1.9) france 1 (1.9) switzerland 1 (1.9) denmark 1 (1.9) total 54 (100) most of the articles were published in uk, specifically 105 (62,5 %) of them. in 2015, there were 19 articles (73,1 %) (table 4). seemedj 2019, vol 3, no. 2 clinical supervision in nursing in journals 53 southeastern european medical journal, 2019; 3(2) table 4. list of countries where articles about cs in nursing were published between 2011 and 2015 country publishing journal number (%) of articles total n (%) p* 2011. 2012. 2013. 2014. 2015. usa 12 (26.1) 7 (23.4) 17 (41.4) 5 (20) 5 (19.2) 46 (27.4) 0.18 uk 31 (67.4) 17 (56.7) 20 (48.8) 18 (72) 19 (73.1) 105 (62.5) 0.19 australia 0 1 (3.3) 2 (4.9) 1 (4) 0 4 (2.4) 0.51 canada 2 (4.3) 1 (3.3) 2 (4.9) 0 0 5 (2.9) 0.67 italy 0 2 (6.7) 0 0 2 (7.7) 4 (2.4) 0.09 slovenia 1 (2.2) 0 0 0 0 1 (0.6) 0.62 france 0 0 0 1 (4) 0 1 (0.6) 0.19 switzerland 0 1 (3.3) 0 0 0 1 (0.6) 0.33 denmark 0 1 (3.3) 0 0 0 1 (0.6) 0.33 total 46 (100) 30 (100) 41 (100) 25 (100) 26 (100) 168 (100) in total, 29 articles about cs in nursing are available in full text to the croatian academic community through the academic and biomedical consortium. ovidsp provides access to the largest number of articles, 18 (62,1 %) of them, while pero alone does not provide access to any article in full form (table 5). table 5. available articles about cs in nursing in full text browser (interface) number (%) of articles in full text only pero 0 only pubmed central 1 (3.5) only ovidsp 18 (62.1) ebscohost, pero 7 (24.1) ovidsp, pero 3 (10.3) total 29 (100) seemedj 2019, vol 3, no. 2 clinical supervision in nursing in journals 54 southeastern european medical journal, 2019; 3(2) discussion after searching for articles about cs, it can be concluded that many articles give a review of that topic, especially of a specific topic within cs, for example cs evaluation (9), the effectiveness of group cs in reducing stress (23), organization of cs in nursing (24), empirical research on cs in psychiatric care (7), concept and definition of cs (25), administrative cs (26). in order to find papers about cs, most of the authors searched databases such as medline (pubmed) and cinahl(7, 27, 15, 23, 26). some of them searched psycinfo (7, 15), medic, linda (26), pre-cinahl, and academic elite (27) as well. it can be seen that authors who searched the ebsco database also searched databases cinahl and psycinfo. for the purposes of this research, apart from the cinahl database, databases health source-consumer edition and health source: nursing/academic edition were searched. database psycinfo was not searched. the most commonly used keyword was “clinical supervision” (27, 15, 23). in one case, “clinical supervision” was combined with the keyword “nurse” (27). keywords “supervision” and “nursing supervisory” were used as well (15). it is interesting that only one author used the mesh term “nursing supervisory” (15). none of them used the mesh term “organization and administration”, which is synonymous with the term “supervision” (28). this research showed that articles about cs were published in a lot of journals (if we consider the number of articles). but if we look closer, it is obvious that more than half of the journals, 29 to be precise, published only one article in a period of five years. the rest of the journals did not publish more than ten articles. the journal "practising midwife” published 20 articles and the “journal of nursing management” published 26 articles. acknowledgement. i would like to thank professor ljiljana pavičić for helping me write this paper. the paper was presented as a master's thesis at the faculty of medicine of j. j. strossmayer university in osijek on september ¬¬11, 2017. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references (1) hornby as, wehmeier s, mcintosh c, turnbull j, ashby m. oxford advanced learner's dictionary.7. ed. uk: oup oxford, 2005. 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(27) butterworth t, bell l, jackson c, pajnkihar m. wicked spell or magic bullet? a review of the clinical supervision literature 20012007. nurse educ today 2008;28:264-72. u.s. national library of medicine. mesh browser. available at: https://meshb.nlm.nih.gov/record/ui?ui=d009 934. date last accessed: 3.6.2017. seemedj 2017, vol 1, no. 2 the quality of life of a patient with colostomy 81 southeastern european medical journal, 2017; 1(2) the quality of life of a patient with colostomy1 valentina plazibat1, nada prlić2, ana kovačević3 1 public health center osijek, osijek, croatia 2 faculty of medicine osijek, department of nursing, medical ethics and palliative medicine, josip juraj strossmayer university of osijek, osijek, croatia 3 clinical hospital osijek, otorhinolaryngology and head and neck surgery clinic, osijek, croatia corresponding author: valentina plazibat, ma in nursing – vplazibat@gmail.com received: august 8, 2017; revised version accepted: november 24, 2017; published: november 24. 2017 keywords: colostomy; quality of life abstract aim: the aim of this research was to examine the quality of life of patients with temporary or permanent colostomy regarding age, gender, marital status and time spent with an ostomy. materials and methods: the study involved 41 members of the ostomy ilco clubs in slavonski brod and osijek as respondents. the standard version of the “quality of life questionnaire for a patient with an ostomy” was used as the research instrument. results: the mean value of the overall scale was 5.4. the respondents rated the physical well-being the highest, a median of 5.9 (interquartile range 4.6 to 7.2), and social well-being the lowest, median 5.0 (interquartile range 4.1 to 6). women gave higher marks for physical, social and spiritual wellbeing, and overall for the scale median of 5.5 (interquartile range 4.9 to 6.1), but with no significant differences compared to men. married respondents ranked psychological, social and spiritual wellbeing higher, with a median of 5.4 (interquartile range 4.7 to 6.0), but with no significant differences compared to those who live alone. physical, psychological and spiritual well-being is somewhat lower in patients under the age of 65, and social well-being is lower in subjects aged 66-75, the median of 4.7 (interquartile range 3.6 to 5.6). correlation between the subscales pertaining to quality of life of the respondents with regard to their age and with regard to living alone (single, divorced or widowed) is significant in terms of the physical (spearman coefficient of correlation  = 0.945, p < 0.001), mental (spearman coefficient of correlation  = 0.943, p = 0.005), social (spearman's correlation coefficient  = 0.829, p = 0.042) and spiritual well-being (spearman coefficient of correlation  = 0.886, p = 0.019). conclusion: quality of life of patients with an ostomy is satisfactory. there are differences in quality of life regarding age, gender, marital status and time spent with an ostomy. there is also a connection between the subscales “quality of life regarding age” and “quality of life regarding marital status” of patients with colostomy. (plazibat v, prlić n, kovačević a. the quality of life of a patient with colostomy. seemedj 2017; 1(2); 81-89) seemedj 2017, vol 1, no. 2 the quality of life of a patient with colostomy 82 southeastern european medical journal, 2017; 1(2) introduction year in, year out, cancer incidence and mortality rate are increasing worldwide. colorectal cancer accounts for more than 9% of the world's cancer incidence. it is the third most common cancer in the world and the fourth most common cause of death (1). globally, colorectal cancer (crc) is the third most common cancer diagnosed in men (after lung cancer and prostate cancer), and the second most commonly diagnosed in women (after breast cancer) (2). carcinoma is the second most significant cause of death in croatia: every fourth resident dies from cancer. compared to other european countries, croatia is a country with a medium incidence but high mortality rate (3). colorectal cancer is usually caused by changes on the colon mucous membrane or on a polyp protruding from a mucous membrane (4). like all cancers, it is most curable if detected at an early stage of the disease. in many cases a surgical operation is performed that ends with the creation of a colostomy on the lower left side of the abdominal wall, but it is also possible to create an ostomy on the right side of the abdominal wall. in most cases, the colostomy is temporary and is removed but sometimes remains a permanent solution in cases of poor operation outcome and metastases (5). the main purpose of creating an ostomy is to release the stool and gas, that is, to keep the continuity of the digestive tract (2). before surgery, the patient often feels anxiety, worry and fear, and this is where a nurse plays a major role. health education always begins with preoperative psychological preparation of the patient, as it allows the nurse to assess the patient's level of knowledge of the illness and operation itself, as well as about their family support, work, hobbies, cultural and spiritual beliefs. the success of mental preparation depends largely on the patients themselves and their acceptance of the new situation. if a patient cannot care for him/herself, the education involves a spouse or a family member (6). hospital discharge planning requires team work of doctors, nurses, enterostomal therapists and dieticians. at the discharge, patients and caregivers are provided with special information on the proper way of treating the ostomy and the surrounding skin, changing ostomy bags and using ostomy aids. discharge information also includes instructions about prevention of possible complications. family members participate in the process of cleaning the wound and skin and in changing ostomy bags so they can do it themselves when the patient returns home. patients also get clear directions about when they need to call a doctor (7). the quality of life of a patient with colostomy depends to a great extent on how well they and their family have adopted procedures important for ostomy care and accepted the new lifestyle (6). although a minor surgery from the medical point of view, the creation of temporary or permanent ostomy greatly reduces the quality of the patient's life (8). colostomy has a significant effect on the life of the patient, regardless of the diagnosis of the disease. cohen et al. emphasize that for patients with cancer, concern about the creation of the ostomy goes beyond all the other patients’ concerns (9). the influence of colostomy on physical, psychological, social and spiritual well-being is not unexpected, but is insufficiently described in the literature. research shows that quality of life is increasingly recognized as an important measure of the outcomes in survivors of large surgical and medical treatments. in patients with colon cancer, various physical problems are present after surgical treatment, such as problems with the stool and urination, and problems with intimacy, which significantly affects the quality of life. evaluating the quality of life is considered crucial for evaluating clinical outcomes after surgical treatment since it considers the patient's perspective in the decision-making process (10). new studies have shown that the ostomy has, as expected, a great influence on the patient's quality of life and a great influence on the patient's daily life. the easiest way to find out how an individual perceives his or her quality of seemedj 2017, vol 1, no. 2 the quality of life of a patient with colostomy 83 southeastern european medical journal, 2017; 1(2) life and how satisfied they are with their everyday life after the creation of an ostomy is to use an interview or a questionnaire that assesses the quality of life (11). the quality of life is multi-dimensional, dynamic, and subjective. it is focused on the patient, involving physical, functional, emotional and social/family well-being. this is why quality of life is important for assessing the effects of disease on individuals, their families and their community (12). many studies have shown a reduction in the quality of life in patients with colostomy. dissatisfaction with preoperative preparation and postoperative care, complications related to ostomy, psychiatric history of the patient, and negative thoughts and beliefs associated with ostomy contribute to the reduction of life quality (13). on the other hand, ostomy gives many people hope, prolongs their life, and enables their activity and realization of life plans. adaptation to a new life situation lasts from several months to two years and is conditional upon a number of factors that include one’s general health but also the level of knowledge and skills that the person possesses. the support of patient’s family and health care professionals, as well as joining support groups where they will be able to talk about difficulties and identify with people who have experienced the same situation, is particularly important. all this enables the patient to restore their everyday activities and social roles as soon as possible (14). the patient's attitude toward their own wellbeing and functional status is an important outcome and the priority of each research. according to the guidelines on care quality provided by the swedish national health and social care board, the patients should be treated respectfully; they must receive satisfactory information and have the opportunity to participate in the decisionmaking process. quality of life is essential for patients with colostomy and the factors that affect it should be enabled. assessing the quality of life of patients with an ostomy will lead to a better understanding of the patients and improve their life quality. psychosocial needs as well as their impact on quality of life have been researched, but a small number of studies point to interventions which could solve problems and meet the needs of patients with a colostomy (1, 10). the main purpose of this study was to examine the quality of life of patients with a temporary or permanent colostomy. specific goals were focused on the following issues: a) to examine whether there is a difference in the quality of life of patients with temporary or permanent colostomy with regard to the respondents’ age; b) to investigate whether there is a difference in the quality of life of patients with temporary or permanent colostomy with regard to the patients’ gender; c) to find out whether there is a difference in the quality of life of patients with temporary or permanent colostomy given the respondents’ marital status, d) to examine whether there is a difference in the quality of life of patients with temporary or permanent colostomy regarding the time spent with a colostomy and e) to find out whether there is a correlation between the “quality of life of patients with colostomy” subscales and their age, gender, marital status and time spent with a colostomy. materials and methods our respondents, a total of 41, were members of ostomy ilco club slavonski brod and ostomy ilco club osijek who have either a temporary or a permanent colostomy. subjects were aged 44 to 74, and there were both male and female respondents. all respondents were regular at their monthly ostomy club meetings. as an instrument of research, an anonymous questionnaire was used, consisting of general demographic data (age, gender, marital status, temporary / permanent colostomy, how long a person has had a colostomy). the questionnaire used to assess the quality of life of a person with a colostomy was the “quality of life questionnaire for a patient with an ostomy”, city of hope and beckman research institute (9, 15, 16). this questionnaire consists of 43 questions evaluating the quality of life using a scale from 0 to 10. the questionnaire is divided into four seemedj 2017, vol 1, no. 2 the quality of life of a patient with colostomy 84 southeastern european medical journal, 2017; 1(2) subscales: “physical well-being” (questions 1 to 11), “psychological well-being” (questions 12 to 24), “social well-being” (questions 25 to 36), and “spiritual well-being” (questions 37 to 43). answers are scored, with 0 being the worst possible result, and 10 the best possible one. however, a few answers are scored in the opposite direction, with 0 being the best and 10 the worst result. particles 11-12, 15, 18-19, 22-30, 32-34, and 37 are scored reversely. the results were obtained by adding points to each particle in the subscale, then dividing the sum by the number of particles in each subscale. the total qol score was obtained by summing the results of all the particles and dividing by the total number. statistical analysis categorical data were represented by absolute and relative frequencies. numerical data were described by the median and the limits of the interquartile range. the normality of the distribution of numeric variables was tested by the shapiro-wilk test. differences in numeric variables by gender and marital status, due to deviations from normal distribution, were tested by the mann-whitney u test. the difference in numeric variables by age groups and duration of the ostomy, due to the deviation from the normal distribution, was tested by the kruskal-wallis test. all p values were two-sided. the level of analysis, the statistical program medcalc (version 16.2.0, medcalc software bvba, ostend, belgium) was used. ethical principles prior to the research, a written consent of the commission for ethical and vocational issues at the j. j. strossmayer university of osijek, faculty of medicine osijek was obtained (class: 60204/16-08/15, no.: 2158-61-07-1683, 10th june 2016.). the approval for the use of the standard questionnaire “quality of life questionnaire for a patient with ostomy” was obtained from the city of hope national medical centre principal, marcie grant. the research was conducted in accordance with ethical principles and human rights in research. results the study included 41 respondents aged 44 to 74 (mean age 66 years, interquartile range 61 to 74 years). out of the total number of respondents, 26 (63%) were male and 15 (37%) were female patients. most of the subjects were married, a total of 35 (85%). there were 6 (15%) patients with a temporary colostomy and 35 (85%) patients with a permanent colostomy. the patients’ time spent with the colostomy was 1 to 360 months (mean 48 months, interquartile range 12 to 102 months). the overall quality of life of the examinees with the colostomy includes physical, psychological, social and spiritual well-being. the full-scale reliability coefficient, cronbach alpha, was 0.957. when it comes to physical well-being, 5 respondents (12.2%), had serious sleep problems, followed by problems with smells – 3 (7.3%) of them, and 2 (4.9%) of them reported problems with diarrhea. problems with itching or pain, diarrhea, gases, leakage of the bag contents, and constipation / absence of stool, were the least apparent. regarding their psychological well-being, 6 (14.6%) respondents expressed fears that their illness would relapse. also, it was difficult for them to adjust to the ostomy, and they reported a feeling of uselessness. some respondents feel embarrassed by having a colostomy, some consider the ostomy hard to look at and take care of, some have memory problems and some of them do not feel happy with their lives as a consequence. regarding overall results pertaining to the social well-being components, privacy in the care of the ostomy is not a problem for most respondents. nevertheless, for ten (24.4%) respondents, their condition is completely stressful for the family; six (14.6%) respondents state that their ostomy is a problem during travel, and five (12.2%) respondents report troubles with sports activities and recreation. as for the results of the participants with respect to seemedj 2017, vol 1, no. 2 the quality of life of a patient with colostomy 85 southeastern european medical journal, 2017; 1(2) the aspects of spiritual well-being, seven respondents (17.1%) find that they have a reason to live, whereas four (9.8%) respondents state that they are completely uncertain about their future. the same number of respondents find prayer and meditation sufficient to satisfy their spiritual needs. in order to determine the differences in the quality of life with a colostomy in terms of age of the respondents, they were divided into three groups: the first group with respondents of up to 65 years of age, the second one with those between 66 and 75 years old, and the third group with respondents older than 76 years of age (table 1). analysis of the impact of time spent with an ostomy on the quality of life with respect to physical well-being, mental well-being, social table 1. rating of subscales and the overall scale of the quality of life with a colostomy by age of respondents median (interquartile range) according to age groups p* up to 65 years (n=18) 66 – 75 years (n=16) 76 and over (n=7) in total (n=41) physical well-being 5.7 (4.6 – 6.8) 6.5 (4.5 – 7.1) 6.7 (4.5 – 8.2) 5.9 (4.6 – 7.2) 0.671 psychological well-being 5.1 (4.1 – 5.8) 5.3 (4.8 – 5.8) 5.8 (5.2 – 6.4) 5.2 (4.8 – 5.8) 0.385 social well-being 5.0 (4.4 – 6.1) 4.7 (3.6 – 5.6) 5.1 (4.3 7) 5.0 (4.1 – 6.0) 0.364 spiritual well-being 4.9 (3.9 – 5.9) 5,5 (4.3 – 6.3) 5 (2.9 – 6.1) 5,3 (4.1 – 6.1) 0.444 overall scale 5.3 (4.7 6) 5.3 (4.8 – 5.9) 5.6 (4.6 7) 5.4 (4.7 – 6.0) 0.727 * kruskal wallis test table 2. rating of subscales and the overall scale of the quality of life by the length of time a person has spent living with a colostomy median (interquartile range) according to the length of time spent with a colostomy p* up to 2 years (n=16) from 3 to 10 years (n=19) for 11 or more years (n=6) in total (n=41) physical well-being 6.1 (4.8 – 7.4) 5.4 (4.3 – 6.7) 7 (5.5 – 7.8) 5.9 (4.6 – 7.2) 0.255 psychological well-being 5.2 (4.4 – 5.7) 5.2 (4.8 – 5.8) 5.5 (5.2 – 5.8) 5.2 (4.8 – 5.8) 0.413 social well-being 5.1 (4.2 – 6.1) 4.8 (3.2 – 5.3) 5.9 (4.8 – 6.8) 5.0 (4.1 – 6.0) 0.110 spiritual well-being 4.7 (4.1 6) 5.4 (4.3 – 6.1) 4.6 (3.6 – 7.3) 5.3 (4.1 – 6.1) 0.657 overall scale 5.3 (4.7 – 6.2) 5.3 (4.4 – 5.6) 5.6 (5.3 – 6.2) 5.4 (4.7 – 6.0) 0.489 * kruskal wallis test seemedj 2017, vol 1, no. 2 the quality of life of a patient with colostomy 86 southeastern european medical journal, 2017; 1(2) well-being, and spiritual well-being, is shown in table 3. rating of subscales and the overall scale of the quality of life of a person with a colostomy median (interquartile range) minimum maximum physical well-being 5.9 (4.6 – 7.2) 1.5 – 9.3 psychological well-being 5.2 (4.8 – 5.8) 3.4 – 7.5 social well-being 5.0 (4.1 – 6.0) 0.9 – 7.4 spiritual well-being 5.3 (4.1 – 6.1) 1.9 – 8.6 overall scale 5.4 (4.7 – 6.0) 3.0 – 7.4 table 4. assessment of the correlation between the age of the subjects and the length of time spent living with a colostomy, according to marital status spearman coefficient of correlation (p-value) age of responden ts length of life with colostomy physical well-being psychologi cal wellbeing social wellbeing spiritual well-being married physical well-being 0.082 (0.638) -0.103 (0.554) psychological wellbeing -0.038 (0.829) 0.112 (0.520) 0.118 (0.500) social well-being -0.139 (0.426) 0.027 (0.879) 0.683 (<0.001) 0.350 (0.040) spiritual well-being -0.036 (0.838) 0.028 (0.874) -0.211 (0.224) 0.137 (0.432) -0.083 (0.637) overall scale -0.140 (0.423) -0.032 (0.856) living alone (single, divorced, widower) physical well-being 0.945 (<0.001) 0.543 (0.266) psychological wellbeing 0.943 (0.005) 0.371 (0.468) 0.943 (0.005) social well-being 0.829 (0.042) 0.543 (0.266) 0.829 (0.042) 0.714 (0.111) spiritual well-being 0.886 (0.019) 0.429 (0.397) 0.886 (0.019) 0.943 (0.005) 0.829 (0.042) overall scale 0.987 (<0.001) 0.543 (0.266) seemedj 2017, vol 1, no. 2 the quality of life of a patient with colostomy 87 southeastern european medical journal, 2017; 1(2) well-being, and spiritual well-being, is shown in table 2. respondents rated physical well-being the highest, with a median of 5.9 (interquartile range 4.6 to 7.2), ranging from 1.5 to 9.3, and gave the lowest scores for social well-being, a median of 5.0 (interquartile range 4.1 to 6), ranging from 0.9 to 7.4 (table 3). there is a significant correlation between the quality of life subscales according to the respondents’ age for those living alone (single, divorced or widowed) in terms of their physical (spearman coefficient of correlation  = 0.945, p < 0.001), mental (spearman coefficient of correlation  = 0.943, p = 0.005), social (spearman's correlation coefficient  = 0.829, p = 0.042) and spiritual well-being (spearman coefficient of correlation  = 0.886, p = 0.019). the overall scale is related to the age of the respondents (spearman's coefficient of correlation  = 0.987, p < 0.001). physical well-being is significantly associated with psychological well-being (spearman coefficient of correlation  = 0.943, p = 0.005), social well-being (spearman correlation coefficient  = 0.829, p = 0.042) and spiritual wellbeing (spearman coefficient of correlation  = 0.886, p = 0.019). spiritual well-being is significantly and strongly associated with psychological (spearman's coefficient of correlation  = 0.943, p = 0.005) and social wellbeing (spearman coefficient of correlation  = 0.829, p = 0.042) (table 4). discussion the overall quality of life of patients with a colostomy involves physical, psychological, social and spiritual well-being. in the context of physical well-being, the results of this study show that respondents are most concerned with sleep problems, smells and diarrhea. they have least problems with itching or pain, gases, leakage of the bag contents, and constipation / absence of stool. in the research conducted by grant et al., the subjects complained most of gas problems, smell, diarrhea and leakage of the bag contents (17). in the study of krouse, herrington et al., it was reported that subjects had the most problems with the skin around the ostomy, with unpleasant smells and leakage of the bag contents (18). in the research conducted by dabirian et al., the respondents complained about the skin around the ostomy, sleeping problems, unpleasant smells and gases (19). it is evident that, in addition to all the physical problems mentioned, all respondents (both in the present study and in other ones), complained about the smell. pittman et al. point out that these problems can lead to psychological and social problems (20). the results of this research with regard to psychological well-being of persons with a colostomy are supported by the results of earlier studies. thus, for example, krouse, grant et al., state that most of the subjects experience problems with anxiety, getting to know new people, and fearing the return of the disease (21). krouse, herrington et al. report that the survey respondents complained most about having problems when they wanted to travel somewhere because they lacked privacy in maintaining the ostomy and therefore felt anxious (18). mitchell et al. find that subjects in their research felt anxious and depressed (22). orbach and tallent state that dissatisfaction with one’s appearance may last for 5 to 10 years after surgery (12). in the context of social well-being, the results of our research show that privacy in maintaining the ostomy is not a problem for most respondents. however, most patients reported that their condition was completely stressful for their family. some of them stated that their ostomies disturbed their intimacy or posed problems in sports and recreation, which is consistent with the results of other studies (17, 18, 20, 21). in research results of mitchell et al. we can find that respondents felt isolated from society (22). the results of this research show that, in the context of spiritual well-being, most respondents feel that they do have a reason to live. some of them are completely uncertain seemedj 2017, vol 1, no. 2 the quality of life of a patient with colostomy 88 southeastern european medical journal, 2017; 1(2) about their future, while others find prayer and meditation to be enough to satisfy their own needs. conversely, in the research by grant et al., the number of respondents who stated that they have a reason to live was the smallest. similar to the findings of our study, respondents in grant et al. research are uncertain about their future, but have religious support, and inner peace (17). in the research of krouse, grant et al., the greatest problems are related to inner peace, reasons for living, and religious support (21). dabirian et al. stated in their findings that the most problematic issue for their respondents was the conduct of religious activities, i.e. going to worship (19). in the research of mitchell et al., patients are least likely to find hope for the future (22). the nurse, as one of the persons who play a role in health education, provides the patient with physical and psychological preparation. in this process, the most important thing is to include family members and spouses. given that both before and after surgery the patients feel anxious, afraid and worried about the outcome, the nurse must have patience and be extremely sympathetic to their health problems, including those pertaining to their physical, psychological, social and spiritual well-being. the nurse also provides support after the setting up of the colostomy, provides patient and family education and psychological support, and helps with the patient’s participation in an ostomy club, so that the patient understands that he is not alone. conclusion quality of life of patients with a colostomy is satisfactory. physical, psychological and spiritual well-being is somewhat lower in subjects aged 65 and under, and social well-being is lower in subjects aged 66 to 75 but with no significant differences in other age groups. women have given somewhat higher grades for their physical, social, and spiritual well-being, and for overall scale, but without any significant differences in relation to men. married respondents have ranked their psychological, social and spiritual well-being as well as the overall scale higher, but without any significant differences in relation to those living alone (single, divorced, widowed). the assessment of physical and social wellbeing is the lowest in patients living with an ostomy for three to ten years. spiritual wellbeing is graded the lowest in respondents living with an ostomy for up to two years, while assessments of psychological well-being are equal according to the time spent living with a colostomy. although present, the differences are not statistically significant. there is also a connection between the subscales “quality of life regarding age” and “quality of life regarding marital status” of patients with an ostomy. acknowledgements i would like to thank all the institutions in which this survey was conducted, as well as all participants in the research, for their helpfulness. the paper was presented as a graduate thesis at the j. j. strossmayer university, faculty of medicine in osijek on september 20, 2016. funding: no specific funding was received for this study. transparency declaration: competing interests: none to declare. references 1. simmons k.l., smith j.a., bobb k.-a, liles l.l.m., adjustment to colostomy: stoma acceptance, stoma care self-efficacy and interpersonal relationships. j adv nurs 2007;60(6), 627–635 2. bazaliński d, sałacińska i, więch p, kózka m. life satisfaction and self-efficacy in patients with stoma. prog health sci 2014;4(2):22-30. 3. stevanović r, capak k, benjak t: statistički ljetopis republike hrvatske 2015. zagreb: državni zavod za statistiku republike hrvatske; 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(whoqol group. the world health organization quality of life assessment (whoqol): position paper from the world health organization. soc sci med 1995; 41: 1403–9.) 18. krouse rs, herrinton lj, grant m, wendel cs, green sb, mohler mj, baldwin cm, mcmullen ck, rawl sm, matayoshi e, coons sj, hornbrook mc. health related quality of life among long-term rectal cancer survivors with an ostomy: manifestations by sex. j clin oncol 2009;27(28):4664-70. doi: 10.1200/jco.2008.20.9502 19. dabirian a, yaghmaei f, rassouli m, tafreshi mz. quality of life in ostomy patients: a qualitative study. patient prefer adherence 2011;5:1-5. 20. pittman j, rawl sm, schmidt cm, grant m, ko cy, wendel c. et al. demographic and clinical factors related to ostomy complications and quality of life in veterans with an ostomy. j wound ostomy continence nurs 2008;35(5):493-503. doi: 10.1097/01.won.0000335961.68113.cb. 21. oecd social expenditure database (socx), 1980–2001. organisation for economic cooperation and development, paris; 2004. 22. mitchell ka, rawl sm, schmidt cm, grant m, ko cy, baldwin cm, wendel c, krouse rs. demographic, clinical, and quality of life variables related to embarrassment in veterans living with an intestinal stoma. j wound ostomy continence nurs 2007;34(5);524-532. seemedj 2017, vol 1, no. 1 etiology and the genetic basis of intellectual disability in the pediatric population 144 southeastern european medical journal, vol 1, 2017. etiology and the genetic basis of intellectual disability in the pediatric population 1 višnja tomac1,2, silvija pušeljić1,2, ivana škrlec3, mirna anđelić3, martina kos1, jasenka wagner3 1 pediatric clinic, clinical hospital centre osijek, osijek, croatia 2 department of pediatrics, faculty of medicine osijek, josip juraj strossmayer university of osijek, osijek, croatia 3 department of medical biology and genetics, faculty of medicine, josip juraj strossmayer university of osijek, osijek, croatia corresponding author: višnja tomac, md visnja.tomac@yahoo.com definition and prevalence of id/mr intellectual disability/ mental retardation (id/mr) is defined as a disability characterized by significant limitations in intellectual functioning and in adaptive behavior; condition covers everyday social and practical skills and begins before the age of 18. intellectual functioning, also called intelligence, refers to received: march 15, 2017; revised version accepted: may 25, 2017; published: june 2. 2017 keywords: mental retardation, intellectual disability, etiology, submicroscopic chromosome aberrations, genes general mental capacity, such as learning, reasoning, problem solving and so on. adaptive behavior is the collection of conceptual (language and literacy), social (interpersonal skills, social responsibility) and practical skills (activities of daily living and personal care, occupational skills, healthcare) that are learned and performed by people in their everyday living (1). abstract intellectual disability/mental retardation (id/mr) is defined as incomplete mental and cognitive development present before the age of 18. there are number of pre-natal and post-natal risk factors that can cause id/mr but 25 %-50 % of all have genetic causes. in the general population, the prevalence of id/mr is about 2 %-3 %. use of standard cytogenetic methods analysis of chromosomes (gtg banding) and fish (fluorescent in situ hybridization) reveals only a small number of causes, but when using new molecular genetics techniques (like chromosomal microarray and next generation sequencing), the rate of causes of id/mr is increased and new candidate genes for id/mr have been discovered. establishing a diagnosis of id/mr is important for the patient and it provides genetic counseling for parents. (tomac v, pušeljić s, škrlec i, anđelić m, kos m, wagner j. etiology and the genetic basis of intellectual disability in the pediatric population. seemedj 2017;1(1);144-153) seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 145 southeastern european medical journal, vol 1, 2017. the intelligence quotient test (iq test) is a major tool in measuring intellectual functioning, which is the mental capacity for learning, reasoning, problem solving and so on. a test score below or around 70 or as high as 75 indicates a limitation in intellectual functioning. iq testing became the way to define groups and classify people within them (1). according to iq testing, id/mr is categorized as: mild (iq 50 55 to 70), moderate (iq 35 49 to 50 55), severe retardation (iq 25 20 to 35-40), or profound retardation (iq below 20). the prevalence of id/mr varies considerably due to the different criteria and methods used in the diagnosis. this problem is present in 2 % to 3 % of the children's population, especially because 5 % to 10 % of children have motor impairment, isolated speech and language delay, severe primary sensorial deficits and pervasive disabilities. id/mr is more frequent in countries of lower socioeconomic status due to increased incidence of anoxia, birth trauma and newborn brain infections (2). the prevalence of mental retardation in developed countries is thought to be 2% to 3%. the prevalence of mild id/mr more often depends on external environmental factors (level of maternal education, access to education, opportunity and access to healthcare), while the prevalence of severe id/mr is relatively stable (3). diagnosis is highly dependent on a comprehensive personal and family medical history, a complete physical examination and a careful developmental assessment of the child. when diagnosing id/mr, it is very important to know how it is defined and classified. etiology and epidemiology of id/mr the etiology of id/mr has heterogeneous environmental and genetic causes, summarized in table 1 (4, 5). prenatal factors are environmental (mother infection in pregnancy such as rubella infections, syphilis, toxoplasmosis, cytomegalovirus and hiv infections), teratogenic (the use of drugs such as thalidomide, phenytoin and warfarin sodium in early pregnancy, radiation), chromosomal abnormalities (e.g. trisomy 21), cryptic chromosomal abnormalities (deletions or duplications) and genetic mutations. perinatal factors are prematurity and asphyxia, while postnatal factors are sepsis, meningitis, encephalitis (commonly caused by hsv 1/2) and various multifactorial causes (poverty and cultural factors). genetic factors are thought to cause id/mr in about 25% to 50% of cases (6). specifically, genetic factors are estimated to be the cause of moderate and severe id/mr (iq<50) in 0.3 % to 0.5 % of cases, of mild id/mr (iq ranging from 50 to 70) in 1 % to 3 % of cases and of severe id/mr in 25 % to 50 % of cases (7). table 1. environmental and genetic causes of intellectual disability prenatal factors perinatal factors postnatal factors genetic: chromosomal abnormalities cryptic chromosome abnormalities deletions/duplications contiguous gene syndromes monogenic diseases prematurity low birth weight asphyxia sepsis/meningitis, encephalitis (hsv 1/2) various multifactorial causes (poverty and cultural factors) environmental: infections (toxoplasmosis, syphilis, rubella, cytomegalovirus and hiv infections) mother disease (diabetes) teratogenic factors (drugs and radiation) metabolic: neonatal hypothyroidism seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 146 southeastern european medical journal, vol 1, 2017. based on the symptoms’ presentation, id/mr is divided into two groups: syndromic and nonsyndromic id/mr. in non-syndromic id/mr, the only pathological manifestation is cognitive deficit, and there are no changes in phenotype and no associated anomalies of organ systems. it can be inherited in three ways: autosomal recessive, autosomal dominant or x-linked mode. syndromic id/mr is related to phenotypic dysmorphy (craniofacial, skeletal), growth changes, neuromuscular changes and metabolic diseases (8). chromosomal abnormalities aberrations in the autosomal chromosome number in live-born babies are restricted to aneuploidies. these abnormalities represent about 10 % of the id/mr that can be detected with conventional cytogenetic methods (9). the majority of cases involve trisomy 21 with a prevalence of 1 to 700, which is clinically expressed as down syndrome (10). other rare chromosomopathies include trisomy 13 (patau syndrome) with a prevalence between 1 in 5,000 and 1 in 29,000 live births (11), trisomy 18 (edwards syndrome) with a prevalence of 1 to 3600 and 1 to 8500 (12), and they are usually lethal in the first week of life. monosomy of any autosomal chromosome is lethal in the earliest stage of embryonic life. there are autosomal structural abnormalities such as wolfhirschhorn syndrome (microdeletion 4p) with a prevalence of 1 to 50,000 (13), cri du chat syndrome (microdeletion 5p) with a prevalence of 1 to 50 000 (14) and sex chromosomal aneuploidies such as klinefelter syndrome (47,xxy) with a prevalence of 1 in 500 to 1,000 newborn males (15). contiguous gene syndromes contiguous gene syndromes are disorders caused by chromosomal abnormalities, such as deletions and duplications, which result in an alteration of normal gene dosage. for most autosomal loci, deletion causes a reduction of gene dosage to structural and functional monosomy. haploinsufficiency for specific genes in the critical interval is implicated for del(7)(q11.23q11.23) in williams syndrome, for del(8)(q24.1q24.1) in langer-giedion syndrome, del(17)(p13.3) in miller-dieker syndrome, and for del(22)(q11.2q11.2) in digeorge syndrome and velocardiofacial syndrome (16). genomic imprinting genomic imprinting is a situation in which there is gene expression from only one of the two alleles inherited from each parent, and it is based on epigenetic modifications of specific allele, such as histone acetylation/methylation and dna methylation (17). the deletion of a chromosome segment containing the active allele of an imprinted gene results in structural monosomy but functional nullisomy (e.g., paternal del(15)(q11.2q13) in prader-willi syndrome and maternal del(15)(q11.2q13) in angelman syndrome). uniparental disomy for the homologue containing the inactive allele results in structural disomy but functional nullisomy (e.g., maternal disomy 15 in prader-willi syndrome and paternal disomy 15 in angelman syndrome). idiopathic id/mr current research has been directed to clarify the genetic base of what was accepted as “'idiopathic id/mr”. the most prevalent structural variations in the human genome are copy number variations (cnvs), which appear predominantly in the subtelomeric regions. genomic variations are a frequent cause of miscarriage, congenital anomalies (ca) and intellectual disability (id) (18). pathogenic cnvs have been detected in 10 % to 15 % of patients with idiopathic id/mr, especially with use of microarray technology. most of cnvs are de novo mutations, but there are also rare inherited mutations with unknown significance (19). over the last few years, cryptic chromosomal anomalies, particularly subtelomeric and interstitial rearrangements (including microdeletions as well as balanced translocations and other chromosomal seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 147 southeastern european medical journal, vol 1, 2017. aberrations) less than 3–5 mb, have emerged as a significant cause of “idiopathic id/mr” (20-22). about half of all segmental aneusomies are found on subtelomeric and terminal regions of chromosomes that are gene-rich, and they are responsible for 5% to 7% of all cases of id/mr (23, 24). monogenic causes x-linked mental retardation (xlmr) is a common cause of monogenic intellectual disability, because most of genes causing id/mr are found on the x chromosome. x-linked forms of mental retardation are estimated to cause 1020% of all inherited cases of id/mr. there is a higher prevalence of id/mr among males relative to females (1.8 in 1000 males; carrier females 2.4 in 1000). however, female carriers may manifest mild symptoms, due to a skewed x-inactivation (25). based on symptoms’ presentation, xlmr can be divided into three groups: 1) syndromes characterized by multiple congenital anomalies (phenotypic dysmorphy, organ anomalies); 2) neuromuscular disorders epilepsy, dystonia, spasticity, muscle weakness and so on without malformations and 3) nonspecific conditions (mrx) – isolated id/mr is the only clinical manifestation. there are 215 xlmr conditions divided according to their clinical presentation: 149 with specific clinical findings, including 98 syndromes and 51 neuromuscular conditions, and 66 nonspecific forms (26). fragile x syndrome (fraxa, omim 309550) is the most common form of syndromic xlmr (20 % of all xlmr cases), with a prevalence of approximately 1:5000 males, and causes intellectual disability in about 1 in 8000 females (27). affected individuals have a folate-sensitive fragile site in the region xq27.3, associated with an expansion of a trinucleotide repeat (cgg) in the 5'-noncoding region of a gene that encodes an rna binding protein termed fmr1. individuals with fragile x syndrome have a lossof-function variant of fmr1 caused by an increased number of cgg trinucleotide repeats (typically >200) accompanied by aberrant cpg methylation of fmr1 (28). another common gene is mecp2 (methyl cpg binding protein 2 (omim 300005) on chromosome xq28, which causes rett syndrome, affecting approximately 1 in every 10,000–15,000 females worldwide (29). but it is also identified in the clinical spectrum seen in males with severe neonatal-onset encephalopathy or with x-linked intellectual disability associated with psychosis, pyramidal signs, parkinsonian features and macroorchidism (ppm-x syndrome; omim 300055) (30). evaluation and testing the clinical geneticist has an important role in the evaluation of patients with intellectual disability and in making decisions about further genetic testing. evaluation includes physical examination and the collection of family history information. the physical exam should focus on dysmorphological and neurological evaluation, congenital malformations, somatometric measurements and behavioral evaluations. in all patients with neurological symptoms, such as epilepsy and macro/microcephaly, neuroimaging studies mri (magnetic resonance imaging) should be performed for evaluation of brain malformations. if there are signs of metabolic disease, metabolic tests should be done (organic acid in urine, amino acids in serum, lactate, pyruvate) (31). when investigating a patient with id/mr, with or without dysmorphic features, the initial analysis several years ago usually began with cytogenetic testing (gtg-banding). gtg banding (g-banding with trypsin/giemsa) is used for the detection of aneuploidy (abnormal number of chromosomes) and the identification of structural aberrations: deletions and translocations in chromosomal rearrangements only larger than 5–10 mb. the overall yield of routine cytogenetic testing is 3.7 % (32). fluorescent in situ hybridization (fish), using location specific probes, detects seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 148 southeastern european medical journal, vol 1, 2017. submicroscopic alterations less than 5 mb that cannot be observed using standard cytogenetic tests (gtg-banding). today this method is used when a specific syndrome is suspected with high frequency in the general population (e.g., digeorge/velocardiofacial syndrome, williams beuren syndrome). the yield of fish screening on patients with moderate to severe id/mr is 6.8 % (33). candidates for subtelomere screening are patients with id/mr and two or more dysmorphic features (mostly facial), congenital organ abnormalities, skeletal abnormalities, positive family history and pre/postnatal poor growth/overgrowth (34). several assays are currently available to detect subtelomeric rearrangements, but subtelomeric fish and subtelomeric mlpa have been the most frequently used. mlpa results needed to be confirmed using other more accurate techniques such as fish or acgh (35). with the introduction of comparative genomic hybridization on microarrays it is possible to screen the entire genome for evaluation of deletions and duplications of specific dna sequences. comparative genomic hybridization on microarrays (array comparative genomic hybridization acgh) and the technical basis of the method was first published in 1997 (36). detection of subtle submicroscopic changes in a number of copies of dna less than 1mb is possible using different platforms. with the application of acgh in patients with id/mr it is possible to determine etiology in 20% of patients with normal karyotype and subtelomere screening with mlpa (36). copy number variations (cnvs) are the most prevalent structural variations in the human genome, which appear largely in the subtelomeric regions and can be detected by acgh. id/mr is associated with variable sizes of cnvs (18). a disadvantage of the acgh is that the identification of de novo cnvs of uncertain significance and unreported cnvs can be challenging to interpret. cnvs should be listed as benign or pathogenic, or reported as variants of unknown clinical significance (37). pathogenic variants are detected in 15 % to 20 % of id/mr patients (37, 38). not all cnvs are fully penetrated or cause a spectrum of phenotypes, including intellectual disability, autism, schizophrenia, and dimorphisms. such cnvs can pose challenges to genetic counseling. more variants of uncertain significance are found with higher density arrays (38). sometimes, variants of unknown significance can be resolved by trio testing (mother, father and proband). interpretation of those variants is very comprehensive and challenging, and demands bioinformatics and clinical knowledge. next-generation sequencing (ngs) is dna sequencing technology that sequences all genes in one genetic test. exome sequencing analyzes all exons of protein coding genes in the genome known as a cause of the diseases (clinical exome sequencing) or all of the genes in the genome (whole genome sequencing). ngs in a clinical setting opens up possibilities for discovering the genetic contribution for a large percentage of id/mr individuals at the first onset of symptoms and the possible opening up of pathways for therapeutic interventions (37). ngs is progressively being set up in clinical laboratories for the diagnosis of id/mr because of a higher diagnostic yield and devaluation in costs. many studies revealed the usefulness of using an integrative approach to examine genotype-phenotype variability (37, 39, 40). whole exome sequencing (wes) is an impressive tool for identifying clinically undefined forms of id/mr, especially when acgh identified a de novo cnv of uncertain significance (37). the vast majority of benign variants are single base pair substitutions. with better coverage depth, wes is adequate for the detection for close to all (99.7 %) pathogenic variants (41). cnv analysis is still an active area of research in ngs variant analysis and has long been important in id research. cgh microarrays can only detect unbalanced structural variants, while apparently balanced chromosomal rearrangements occur in 1.54 % of live births and contribute to 6 % of abnormal phenotypes including id (42). whole seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 149 southeastern european medical journal, vol 1, 2017. genome sequencing (wgs) has the potential to uncover all forms of genetic variation in one test, and offers a higher diagnostic yield (43). in the study of harripaul et al. of patients with severe id, a diagnostic yield of 42 % was observed (42) which is a significant improvement over the diagnostic yield obtained by microarray, gene panels or wes (44). a summary of genetic methods used in diagnosing id/mr is presented in table 2. discussion defining the cause of intellectual disability/mental retardation (id/mr) presents a diagnostic challenge. mental retardation is present in about 1 % to 3 % of individuals in the general population, but there are many cases that cannot be explained despite novel technology and clinical investigations (24). genetic factors are involved in many of the idiopathic cases of id/mr. this conclusion is based on the fact that these patients often show signs such as dysmorphic features, growth retardation and malformations, or have a family history of mental retardation (6,7). the genetic heterogeneity of intellectual disability requires genome wide approaches, including the detection of chromosomal aberrations by chromosomal microarrays and whole exome sequencing adequate for discovering single gene mutations (45). for individuals with idiopathic id/mr, autism spectrum disorders, or multiple congenital anomalies, chromosomal microarray analysis (cma) is recommended as the first-line diagnostic test since it offers a much higher diagnostic yield (15 % to 20 %) compared with gbanded karyotype analysis (3 %) (38,42). despite those modern technologies, the genetic etiology of 80 % to 85 % of patients still remains unknown. ngs-based testing (targeted table 2. advantages and disadvantages of genetic methods for diagnosing intellectual disability method advantages disadvantages gtg (g banding with trypsin and giemsa) whole genome analysis detection of unbalanced and apparently balanced chromosomal rearrangements time consuming small resolution (5 to 10 mb) fish (fluorescent in situ hybridization) detection of unbalanced and apparently balanced chromosomal rearrangements and mosaicism detection of small deletions and duplications time consuming small resolution (depend on the size of fish probe, 30 to 100 kb) mlpa (multiplex ligation probe amplification) high-throughput simultaneously analyses of several samples multiplex technique (study of several regions of the human genome in a single reaction) low cost not whole genome analysis sensitive to pcr inhibitors acgh (microarray comparative genomic hybridization) whole genome analysis high resolution (up to 40 kb) impossibility of detection of apparently balanced chromosomal rearrangements and mosaicism cnvs of unknown significance in clinic ngs (next generation sequencing) whole genome analysis high resolution (covering all coding variation) single strand sequencing cnvs of unknown significance in clinic expensive seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 150 southeastern european medical journal, vol 1, 2017. multigene panels, whole exome sequencing or whole genome sequencing) for these cases, has a great potential to obtain diagnosis (44). the vast majority of individuals with id/mr currently receive no molecular diagnosis, which is a shortcoming that significantly impacts health and life span. there is also a strongly negative correlation of survival with the severity of id (46). it is important to emphasize that knowing which genes carry mutations that cause id/mr can have huge benefits for diagnosis in clinics, and can lead to better understanding of each patient’s health issues, more appropriate care and treatment, improved overall health and life span, and appropriate counseling and planning for families. abbreviations id/mr intellectual disability/mental retardation; gtgg-banding with trypsin/giemsa; fish fluorescent in situ hybridization; mlpa-multiplex ligation dependant probe amplification; acgh array comparative genomic hybridization; ngs – next generation sequencing; cnvs copy number variations; xlmr – x linked mental retardation; xlid – x linked intellectual disability; iqintelligence quotient test; fraxa-fragile x syndrome; mri magnetic resonance imaging; ca congenital anomalies. funding this work was supported by the scientific project vif2015-mefos-11 ‘etiology of mental retardation in pediatric population’ faculty of medicine, j. j. strossmayer university in osijek transparency declaration competing interests: none to declare references 1. aaid-resources for intellectual and developmental disability professionals internet.aaidd.org.2017. available from: https://aaidd.org (accessed on 6th may 2017) 2. drews cd, yeargin-allsopp m, decoufle p, murphy cc. variation in the influence of selected sociodemographic risk factors for mental retardation. am j public health 1995;85(3):329–34. 3. leonard h, wen x. the epidemiology of mental retardation: challenges and opportunities in the new millennium. ment 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loreta c, tautvydas r, et al. identification of genetic causes of congenital neurodevelopmental disorders using genome wide molecular technologies. acta med litu 2016;23(2):7385. seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 153 southeastern european medical journal, vol 1, 2017. 46. bittles ah, petterson ba, sullivan sg, hussain r, glasson ej, montgomery pd. the influence of intellectual disability on life expectancy. j gerontol a biol sci med sci 2002;57(7):m470-2. seemedj 2022, vol 6, no 2 fractionation of vipera berus berus snake venom 20 southeastern european medical journal, 2022; 6(2) original article fractionation of vipera berus berus snake venom and detection of bioactive compounds targeted to blood coagulation system 1 eldar iskandarov 1,2*, volodymyr gryshchuk 1, oleh platonov 1, yevhenii kucheriavyi 1, oleksandr slominskyi 1, yevhenii stohnii 1, volodymyr vartanov 1, volodymyr chernyshenko 1 1 palladin institute of biochemistry, nas of ukraine, ukraine 2 biology and medicine institute science educational center of taras shevchenko national university of kyiv, ukraine *corresponding author: eldar iskandarov, iskandarov.e.sh@gmail.com received: sep 21, 2022; revised version accepted: oct 24, 2022; published: nov 28, 2022 keywords: snake venoms, blood coagulation, fibrinogen, hemolysis, platelet aggregation abstract introduction. the performed research focused on a search for new biologically active compounds acting on blood coagulation system proteins and cells. to achieve this goal, we fractionated vipera berus berus snake venom and studied the action of the separated fractions on human blood plasma, fibrinogen, platelets or red cells. methods. crude venom was fractionated using ion-exchange chromatography. protein composition of fractions was studied using sds-page. the ability of fractions to prolong or initiate blood plasma clotting was studied using the prothrombin time test with thromboplastin. fibrinogen-specific proteases were detected using enzyme-electrophoresis. action on red cells was estimated using the hemolysis test. aggregometry was used for the detection of action on platelets. all experiments in this study were performed in vitro. results. we obtained fractions containing phospholipase and a protease that is able to hydrolyze fibrinogen, leading to the loss of its ability to polymerize and to maintain platelet aggregation. conclusion. further purification and study of these components can be a promising research direction for biotechnological as well as for biomedical use. (iskandarov e*, gryshchuk v, platonov o, kucheriavyi y, slominskyi o, stohnii y, vartanov v, chernyshenko v. fractionation of vipera berus berus snake venom and detection of bioactive compounds targeted to blood coagulation system. seemedj 2022; 6(2); 20-31) seemedj 2022, vol 6, no 2 fractionation of vipera berus berus snake venom 21 southeastern european medical journal, 2022; 6(2) introduction snake venoms are complex mixtures of bioactive components, particularly proteins, nucleic acids, and organic compounds. the venom’s main purpose is to immobilize or kill prey, usually small mammals, reptiles, birds or amphibians. the venom’s components get into the prey’s blood during the bite, and are instantly distributed through its body via blood flow. evolutionary, venomous reptiles have developed different routes to hit the prey, one or a few of which appeared to be more successful and thus started to prevail. most snake venoms have neurotoxic action [1]; however, some snakes kill their prey by inducing generalized inflammatory tissue response, or by using blood coagulation activators, which cause massive coagulation or conversely cause hemolysis, inability of the blood to clot, destroy blood vessels and lead to internal hemorrhages [2]. in any case, if one way of immobilizing a preferable type of prey prevails, other venom components do not vanish from the venom instantly. instead, their effects are masked by the more active components. this is why venoms of some snakes simultaneously contain, for instance, enzymes hydrolyzing fibrinogen, which decrease blood coagulation ability, and fibrinogen activators, which stimulate thrombus production, in different proportions. since venom comes into contact primarily with the prey’s blood, the action of many of its components is aimed at the circulatory system. in particular, the proteases destroying fibrinogen, platelet aggregation inhibitors, activators of blood coagulation factors, phospholipases, etc. were found in snake venoms [2]. obtaining and describing these components led to the discovery of unique enzymes, used nowadays in medicine and laboratory diagnostics. that is why the study of snake venom composition is a current issue of modern biotechnology [3]. functionally active components of snake venoms may also be used in fundamental research on the features of certain elements of the hemostasis system. in particular, fibrinogenases (enzymes capable of selectively hydrolyzing fibrinogen) appear to be useful tools for studying the role of certain molecule sites [4]. in our research, we focused on the venom of a snake from ukrainian fauna, which made our object available for analysis, as well as for potential further biotechnological production. the purpose of the study was to search for vipera berus berus venom proteins, which can be used as effectors of the blood coagulation system. materials and methods materials a) vipera berus berus venom crystalline venom of vipera berus berus was provided by the laboratory of experimental herpetology of the trypillia biochemical factory (trypillia, ukraine). b) human blood plasma blood was taken from the vein using a sharp dry needle with a large diameter without a syringe (to prevent hemolysis), discarding the first 5–6 blood drops. during blood taking, neither a tourniquet nor a massage was applied in order to prevent blood coagulation activation and fibrinolysis. blood collection was performed under fasting conditions. a 3.8% sodium citrate solution was introduced into the polyethylene tube using a dispenser and further admixed with blood in a 1:9 ratio. next, the tube was tightly closed and gently mixed using slow hand movements, without shaking. platelet-rich plasma (prp) was obtained by centrifugation at 200 g for 30 min [5]. during centrifugation, the tubes were open so as not to prevent oxygen access to the platelets. seemedj 2022, vol 6, no 2 fractionation of vipera berus berus snake venom 22 southeastern european medical journal, 2022; 6(2) platelet-poor plasma was obtained by centrifugation at 450 g for 20 min at a temperature lower than 20 °с. this study was approved by the ethical committee of the palladin institute of biochemistry of nas of ukraine, on 9 december 2021, n12. volunteers signed informed consent forms prior to blood sampling, in accordance with the declaration of helsinki. c) human fibrinogen fibrinogen was obtained from human blood plasma by salt extraction using 16% na2so4. the content of protein coagulated by thrombin was 96–98%. to remove vitamin k dependent proteins – prothrombin, factors ix and x of the blood coagulation system, and protein c – plasma was cooled to +4 °с and, under constant mixing, admixed with baso4 (30 g/l of blood), then centrifuged at 1300 g for 10 min. the baso4 addition procedure was repeated twice. in the next step, blood plasma was heated to room temperature, and 1 m glycine buffer (glycine and 1 m naoh, ph 9) was added at a 1:10 ratio to the entire volume. after that, one fraction was precipitated using 16% na2so4 (slowly adding a small portion thereof with constant mixing). the resulted precipitate was spundown at 1300 g for 30 min at ambient temperature. supernatant was carefully discarded, and then fibrinogen precipitation was performed using 16% na2so4. after that, fibrinogen was spun-down for 30 min at 1300 g at a temperature of 10–15 °с. the precipitate was then diluted in 0.2 m nacl – 250 ml of solvent was used for solving the precipitate from 1 l blood plasma. the obtained fibrinogen was re-precipitated by an equal volume of 16% na2so4, and then the precipitate was diluted in 0.2 m nacl (150–200 ml for 1 l blood plasma). after that, the fibrinogen solution was added with 0.5 m phosphate buffer (kh2po4 and naoh, ph 6.5) at a 1:5 ratio to the entire fibrinogen solution volume, and then again re-precipitated by equal volume of 16% na2so4. the order of steps is analogous with the first precipitation, but re precipitated fibrinogen was diluted with 0.15 m nacl (70–150 ml of solvent for 1 l blood plasma). re-precipitated fibrinogen was placed on ice with a temperature of +4 °с for the night in order to separate the cryoforms of fibrinogen. the expected concentration of fibrinogen while cryoforms were separating was 10–12.5 mg/ml. then, the fibrinogen solution was centrifuged for 30 min at 1300 g, the supernatant liquid was carefully discarded, and another re precipitation by 16% na2so4 was performed. the precipitated fibrinogen was diluted in 0.15 m nacl, after which the solution was frozen and stored at -20 °с [6]. d) reagents the following materials and reagents were used in the present experiments: acrylamide, bisacrylamide, molecular mass markers for electrophoresis (fermentas, eu), tris (merck, usa), sds (bio-rad laboratories), q sepharose (pharmacia, sweden), pd-10 columns. the thromboplastin (thromborel) was bought from siemens, germany. molecular weight markers sm0671 (250; 130; 100; 70; 55; 35; 25 та 10 kda) were purchased from fermentas (eu). blueye molecular weight markers (245; 180; 100; 75; 63; 48; 35; 25; 20; 17 та 11 kda) were purchased from sigma-aldrich (usa). methods a) ion-exchange chromatography q sepharose is the anion-exchange agent, which allows efficient fractioning of the protein mixture under alkaline ph [7]. to fraction the venom, q sepharose was balanced with 0.05 m tris-hcl buffer, ph 8.9. the column volume was 12.5 ml, and flow speed was 1 ml/min. the adsorbed proteins were eluted with the same buffer in staged gradient: 0.1, 0.2, 0.5 and 2 m nacl, ph 8.9. for fractioning, the acta prime (acta, usa) chromatographic system was used. the obtained material was described using sdspage. seemedj 2022, vol 6, no 2 fractionation of vipera berus berus snake venom 23 southeastern european medical journal, 2022; 6(2) b) protein concentration identification protein concentration in obtained fractions was approximately identified through spectrophotometry, by optic absorbency under 280 nm subtracting the absorbency under 320 nm using the optizen pop (optizen, korea) spectrophotometer. the bradford method was used for quantitative identification of protein concentrations [8]. c) electrophoresis in polyacrylamide gel electrophoresis in polyacrylamide gel (page) with sds was performed according to the laemmli method, where the tris-glycine system is used in the device for vertical gel electrophoresis in the plates [9]. samples for electrophoresis were prepared in accordance with the following algorithm: the 1 mg/ml protein solution was added with a sample buffer that contained 5% sucrose or glycerin, 2% sds and bromophenol (bromophenol is added until coloration appearance). to identify the sites containing proteins, gelplates were developed by staining it in a solution (0.125% coomassie g-250 in 25% isopropanol and 10% acetic acid) for 10 minutes. to discard the staining remains, we used a 3–9% acetic acid solution. the method resolution was 1 µg of protein. to establish the molecular mass of studied proteins, molecular markers were used (fermentas, eu): sm0671 (250; 130; 100; 70; 55; 35; 25 та 10 kda). d) enzyme-electrophoresis with fibrinogen as substrate enzyme-electrophoresis was performed using the laemmli method in 15% paag [9]. the 0.5 mg/ml fibrinogen was put into the separating gel before polymerization. after electrophoresis, sds was removed from the gel through trice washing with 2.5% triton x-100 for 30 min. the gel was incubated in 0.1 m glycine buffer, ph = 8.3, for 12 hours. after that, the gel was stained with coomassie g-250, and the sites of proteolytic activity were identified by uncolored spots on the gel [9]. to establish the molecular mass of studied proteins, molecular markers were used (sigmaaldrich, usa): blueye (245; 180; 100; 75; 63; 48; 35; 25; 20; 17 та 11 kda). e) prothrombin time (pt) this method consists of measuring the coagulation time of citrate plasma after thromboplastin and ca2+ addition. normal prothrombin time is 12–17 s. elongation of prothrombin time may be linked to deterioration of vitamin k-dependent factor synthesis, which are involved in the external pathway of blood coagulation – vii, x, ii – and also to indirect anticoagulant therapy. shortening of prothrombin time is linked to anticoagulant content decrease in the blood, or to pathological activation of the procoagulant chain of the hemostasis system [10]. to perform the test, 80 µl of blood plasma was added with 20 µl of examined fraction (or equal volume of 0.05 m tris-hcl buffer, ph 8.9, in control), and with 100 µl of 0.025 m cacl2 solution, and then polymerization was initiated by adding 100 µl of thromboplastin (thromborel, siemens, germany). the time of clot formation was controlled. f) establishing hemolytic activity red blood cells were obtained from citrate blood via centrifugation for 20 min at 125 g and a temperature of 20 °с. the precipitated erythrocytes were washed by trice resuspending in 10 mm hepes buffer, ph 7.4, containing 0.15 m nacl, 5 mm kcl, 1 mm mgso4, and 10 mm sucrose, with following precipitation at 125 g and a temperature of 20 °с. the suspension of washed human red blood cells obtained using this method [11] was diluted in a 1:8 ratio with 10 mm hepes buffer, ph 7.4, containing 0.15 m nacl, 5 mm kcl, 1 mm mgso4, and 10 mm sucrose. the amount of 50 μl of the suspension was added with the studied sample (total volume – 1 ml), mixed, and incubated at 37 °с for 30 min. seemedj 2022, vol 6, no 2 fractionation of vipera berus berus snake venom 24 southeastern european medical journal, 2022; 6(2) after incubation, the red blood cells were precipitated through centrifugation at 1000 g for 10 min and examined the supernatant’s absorbency at 543 nm against the buffer. hemolysis caused by 50 μl of 1% triton-x100 was treated as 100%. absorbance was measured using the optizen pop spectrophotometer (korea). g) platelet aggregation study platelet aggregation was studied using the solar ар2110 aggregometer (belarus). the 0.2 ml blood platelet-rich plasma (prp) was put into the spectrophotometer cuvette and added with 50 μl of studied fraction, or the equal volume of 0.05 m tris-hcl buffer, ph 8.9, in control. the mixture was incubated for 3 min, then 0.025 ml of 0.025 m cacl2 was added and it was again incubated in the cuvette for 3 min. after that, 0.025 ml of 25 μм adp was added to the cuvette [12]. the entire aggregation process was recorded, and the aggregation level was estimated. h) statistical analysis statistical analysis was performed using microsoft excel (microsoft package). all analyses were conducted in the series of three replicates; standard deviation was considered in data analysis. student’s t-test was used. results are presented as mean ± standard deviation. data were considered significant when p < 0.05. results fractionation of v. berus berus venom for fractionation, 50 mg of crude vipera berus berus venom was diluted in 1 ml of 0.05 m trishcl buffer, ph 7.4. before injection, the venom solution was centrifuged at 200 g for 15 min. using the akta prime chromatographic system, the venom was fractionated at a speed of 4 ml/min using the column, filled with chromatographic sorbent q sepharose of 15 ml volume. after we collected the fraction, which did not bond with the sorbent under present conditions, the column was washed with 0.05 m tris-hcl buffer, ph 7.4. elution was performed using stepping gradient of nacl: 0.1, 0.2, 0.3, 0.5 m of nacl in 0.05 m tris-hcl buffer, ph 8.9. finally, the sorbent was washed with a buffer of 1 m nacl. the chromatogram is presented in figure 1. figure 1. separation of crude vipera berus berus venom on q sepharose. speed of elution was 4 ml/min (elution by stepping gradient of nacl (0.1, 0.2, 0.3, 0.5, 1 m) in 0.05 m tris-hcl buffer, ph = 7.4). seemedj 2022, vol 6, no 2 fractionation of vipera berus berus snake venom 25 southeastern european medical journal, 2022; 6(2) the obtained fractions were de-salted and concentrated using centrifuge microconcentrators amicon ultra 3k. for detection of fractions’ protein composition, gelelectrophoresis was used. sds-page it was demonstrated that all obtained fractions had different protein compositions and contained proteins of molecular mass from 120 to 10 kda (figure 2). this was important for describing each fraction, and for revealing their potential unique physiological effects. a. b. figure 2. a. – electrophoregram of crude vipera berus berus venom. b. – electrophoregram of vipera berus berus venom fractions obtained on q sepharose. m – molecular mass markers (250-10 kda). nb – fraction which did not bind to the sorbent under present conditions. 0.1, 0.2, 0.3, 0.5 – fractions eluted by 0.1, 0.2, 0.3, 0.5 m nacl, respectively. fractions’ impact on fibrinogen and fibrin polymerization to estimate the ability of obtained fractions to inhibit fibrin polymerization, we detected the coagulation time prolongation for blood plasma in the prothrombin time test in the presence of studied fractions. for this purpose, 80 µl of blood plasma was incubated with 20 µl of the fraction at the final concentration of 0.05 o.u./ml (or equal quantity of buffer) for 5 min. the coagulation was initiated by adding 100 µl of 0.025 m cacl2, and 100 µl of thromboplastin solution. it showed prolongation of the coagulation time for blood plasma of up to 60 s (compared to 40 s in control) in the presence of the fraction eluted by 0.2 m nacl. such prolongation may be linked to the presence of the enzyme which can hydrolyze fibrinogen, and which was found in this fraction and in the crude venom. other fractions did not have such ability (figure 3). identification of proteolytic action toward fibrinogen in the chosen fraction was performed using enzyme-electrophoresis with fibrinogen as the substrate. the enzyme-electrophoresis method combines electrophoretic separation and enzymography. in particular, it was proved that the fraction eluted by 0.2 m nacl contained the enzyme capable of hydrolyzing fibrinogen. its approximate molecular mass was 50 kda (figure 4). we concluded that the presence of this enzyme explains the ability of this fraction to slow down fibrin polymerization. seemedj 2022, vol 6, no 2 fractionation of vipera berus berus snake venom 26 southeastern european medical journal, 2022; 6(2) figure 3. time of thromboplastin-induced blood plasma coagulation after 5 minutes of incubation with vipera berus berus venom fractions, obtained on q sepharose. nb – fraction which did not bind to the sorbent under present conditions. 0.1, 0.2, 0.3, 0.5 – fractions eluted by 0.1, 0.2, 0.3, 0.5 m nacl, respectively. * – the result is significant in comparison to control at p < 0.05. the diagram is based on the results of three independent experiments. figure 4. the enzymogram of vipera berus berus venom fraction obtained on q sepharose and eluted with 0.2 m nacl. m – molecular mass markers (245-11 kda); 0.2 – fraction. hemolytic activity to reveal phospholipases in the obtained fractions, their ability to provoke red blood cell hemolysis was studied. for this purpose, a homogeneous suspension of human red blood cells was prepared. as the positive control, the hemolytic agent triton x-100 was used. the samples were incubated at 37 °с for 60 min, then the red blood cells were precipitated by centrifugation, and relative hemolytic activity was estimated by absorbance under 543 nm. red blood cell hemolysis was observed in the presence of the vipera berus berus venom fraction eluted by 0.5 m nacl. it is curious that the gel-electrophoretic data showed the presence of a single protein component of 70 kda mass in this fraction. seemedj 2022, vol 6, no 2 fractionation of vipera berus berus snake venom 27 southeastern european medical journal, 2022; 6(2) figure 5. time of erythrocyte hemolysis under previous incubation with vipera berus berus venom fractions. extinction of supernatant after hemolysis was estimated under the 543 nm light wave. as the control, extinction of supernatant of erythrocyte solution with pbs buffer was taken. 0.5% twin100 – erythrocyte solution with 0.5% twin-100 detergent (positive control). nb – fraction which did not bind to the sorbent under present conditions. 0.1, 0.2, 0.3, 0.5 – fractions eluted by 0.1, 0.2, 0.3, 0.5 m nacl, respectively. * – the result is significant at p < 0.05. the diagram is based on the results of three independent experiments. impact on platelets to estimate the impact of obtained fractions on hemostasis, we used the original approach with modified aggregometry, which allowed us to consider the ability of fractions to activate the platelets, initiate blood coagulation, or inhibit platelet aggregation. for this purpose, 50 ml of the fraction (in the final quantity of 0.05 o.u./ml) was added to 200 µl of platelet-rich blood plasma and incubated in the aggregometer cuvette for 3 min, recording the platelet aggregation process. after that, 25 µl of 0.025 m cacl2 was added, and recording was continued for another 3 min. then, 25 µl of 25 µm adp was added, and platelet aggregation was recorded. decrease of the platelet aggregation level at this stage of measurement may indicate inhibition of aggregation or the damaging of platelets after contact with the studied fractions. the analysis of aggregation curves presented in figure 6 allowed us to conclude that the fraction, which did not bond to the sorbent, and the fraction eluted by 0.1 m nacl were able to activate the platelets moderately. at the same time, the platelet aggregation inhibitor was found in the fraction eluted with 0.2 m nacl. the adp addition did not cause platelet aggregation. it is important to underline that the same fraction had the fibrinogen-specific protease, according to our data. fibrinogen hydrolysis may be one of the causes of inhibition of platelet aggregation, since this process is mediated by fibrinogen. the fraction eluted with 0.5 m nacl did not have an intense impact on the platelets. apparently, 6 minutes of incubation of fractions with platelets was not enough for a manifestation of phospholipase activity, which was predicted by the results of red blood cell hemolysis (60 min incubation). seemedj 2022, vol 6, no 2 fractionation of vipera berus berus snake venom 28 southeastern european medical journal, 2022; 6(2) figure 6. the plot of platelet aggregation in platelet-rich blood plasma after incubation with vipera berus berus venom fractions. control – under the equal buffer volume. nb – fraction which did not bind to the sorbent under present conditions. 0.1, 0.2, 0.3, 0.5 – fractions eluted by 0.1, 0.2, 0.3, 0.5 m nacl, respectively. typical curves for three independent experiments are shown. black lines – points of addition of 25 mkl of 0.025 m cacl2; grey lines – points of addition of 25 µl of 25 µm adp. discussion the fractioning of vipera berus berus venom allowed us to obtain separate fractions, which differ in their protein compositions and in their action on the hemostasis system. in particular, we obtained the fractions containing phospholipase. we also found a protease capable of hydrolyzing fibrinogen, leading to the seemedj 2022, vol 6, no 2 fractionation of vipera berus berus snake venom 29 southeastern european medical journal, 2022; 6(2) loss of its ability to maintain platelet aggregation. the presence of components capable of moderately activating the platelets is expected for some venom fractions. further purification and study of these components may represent a promising research direction. phospholipases, the enzymes that selectively destroy phospholipids, have been obtained from a few snake venoms. in particular, the phospholipases of 12.5, 13.9 and 14.2 kda were obtained from the crotalus molossus nigrescens venom. authors suggest that they should be used in oncological medicine due to their potential as anticancer agents and their lower neurotoxic effects compared to conventional drugs [13]. phospholipases were also found in the naja sumatrana and bothrops alternatus venoms, and methods for their purification were suggested [14,15]. we expect that the application of newly purified phospholipase may be useful for researching the structural-functional features of lipid membranes. recent studies have discussed the practical application of phospholipases. in particular, by inducing treg cells, these enzymes can be effective as potential therapeutic agents in atherosclerosis [16]. antibacterial activity is another promising feature of phospholipases [17,18]. targeting the cell membrane, phospholipases can disrupt the cellular integrity of bacteria. in addition to their potential biomedical use, phospholipases can be applied in studying liposomes and lipid rafts [19]. further study of the fibrinogen-specific protease from the vipera berus berus venom will let us establish its specificity concerning certain chains of the fibrinogen molecule. enzymes capable of selectively hydrolyzing fibrinogen are used in studying its structure and function [20] and obtaining partly hydrolyzed fibrinogen forms [21]. the possibility of fibrinogenase use for thrombolysis is also being discussed [22]. for this purpose, several recombinant analogs for certain fibrinogenases were obtained [23,24]. the mechanism of anticoagulant action of fibrinogen-specific proteases is obvious – cleaving of fibrinogen chains by these enzymes decreases its ability to polymerize [25]. in contrast to other anticoagulants that inhibit coagulation factors (mainly thrombin or factor xa), fibrinogen-specific proteases act in the final step of the clotting process, preventing the formation of a clot [26]. likewise, these enzymes can often effectively cleave polymerized fibrin, thus promoting the dissolution of intravascular thrombus [27]. cardiovascular and renal disease in non-diabetic individuals with arterial hypertension remains a question to be answered. conclusion proteins from vipers’ venoms do not only cause death; they can also be used for treatment of thrombosis, arthritis, cancer and many other diseases [ ]. as snake venom contains a wide spectrum of biological compounds, research conducted for the purpose of identifying their structure, biological activity, and pharmacoclinical application is now of great significance [28]. we have developed a novel method of fractionation of vipera berus berus venom, which enabled us to obtain fractions with different action on the human blood coagulation system. our data corresponded to the recently summarized results presented in [2]. further purification of bioactive proteins that we found in vipera berus berus venom will allow the performance of their full characterization. these molecular effectors can be used for studying regulatory mechanisms of blood coagulation system regulation. acknowledgement. none. disclosure funding. this research was carried out within the framework of the basic theme of the palladin institute of biochemistry of the national academy of sciences of ukraine № 0119u002512 «interactions of the components of the hemostasis system at the cellular and molecular level in the process of thrombus formation and elimination» (2019–2023). competing interests. none to declare. seemedj 2022, vol 6, no 2 fractionation of vipera berus berus snake venom 30 southeastern european medical journal, 2022; 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1148:345-381. 23. he j, chen s, gu j. identification and characterization of harobin, a novel fibrino(geno)lytic serine protease from a sea snake (lapemis hardwickii). febs lett. 2007; 581(16):2965-73. 24. wagstaff aj, gillis jc, goa kl. alteplase. a reappraisal of its pharmacology and therapeutic use in vascular disorders other than acute myocardial infarction. drugs 1995; 50(2):289-316. 25. gardiner ee, andrews rk. the cut of the clot(h): snake venom fibrinogenases as therapeutic agents. j thromb haemost. 2008; 6(8):1360-2. doi: 10.1111/j.15387836.2008.03057.x. 26. chernyshenko v. o, lugovska n. e. molecular mechanisms of intravascular inhibition and stimulation of extravascular thrombosis. biotechnologia acta. 2021, 14(6), pp. 5‒22. https://doi.org/10.15407/biotech14.06.0 05 27. altaf f, wu s, kasim v. role of fibrinolytic enzymes in anti-thrombosis therapy. front mol biosci. 2021; 8:680397. doi: 10.3389/fmolb.2021.680397. 28. pal sk, gomes a, dasgupta sc, gomes a. snake venom as therapeutic agents: from toxin to drug development. indian j exp biol. 2002; 40(12):1353-8. 29. cristina rt, kocsis r, tulcan c, alexa e, boldura om, hulea ci, dumitrescu e, radulov i, muselin f. protein structure of the venom in nine species of snake: from bio-compounds to possible healing agents. braz j med biol res. 2020; 53(1):e9001. author contribution. acquisition of data: ei, vg, op, yk, os, ys, vv, vc administrative, technical or logistic support: ei, vg, op, yk, os, ys, vv, vc analysis and interpretation of data: ei, vg, op, yk, os, ys, vv, vc conception and design: ei, vg, op, yk, os, ys, vv, vc critical revision of the article for important intellectual content: ei, vg, op, yk, os, ys, vv, vc drafting of the article: ei, vg, op, yk, os, ys, vc final approval of the article: ei, vg, os, ys, vc guarantor of the study: vc obtaining funding: os, vv, vc provision of study materials or patients: ei, vg, op, yk, os, ys, vv, vc statistical expertise: ei, vg, op, os, ys, vv, vc seemedj 2017, vol 1, no. 2 thyroid autoimmunity and infertility 1 southeastern european medical journal, 2017; 1(2) thyroid autoimmunity and infertility marin kuharić1, damir rozić2, ivan karner1 1 department of pathophysiology, faculty of medicine osijek, josip juraj strossmayer university of osijek, faculty of dental medicine and health, josip juraj strossmayer university of osijek 2 department of nuclear medicine, university clinical hospital mostar corresponding author: ivan karner md, phd ikarner@mefos.hr introduction the thyroid hormone plays an essential role in the growth, differentiation and development of virtually all tissues. it also impacts numerous metabolic pathways, making it a significant factor in maintaining metabolic homeostasis. the two forms of thyroid hormone, triiodothyronine (t3), which is more potent, and thyroxine (t4), which is more abundant, can have effects on many different cellular components (1). however, they mostly regulate the transcription of target genes by binding to their specific intranuclear receptors, found in several different isoforms (1, 2). thyroid hormones also affect the female reproductive system, which is why dysfunction of the thyroid gland can subsequently lead to disturbances of the menstrual cycle and fertility issues (2). thyroid dysfunction, which leads to changes in circulating thyroid hormone levels, manifests itself as either hyperthyroidism or hypothyroidism. hypothyroidism is the more prevalent variant and is linked to thyroid autoimmunity. autoimmune thyroid disease (aitd) is the most prevalent cause of hypothyroidism in women of reproductive age (3, 4). elevated levels of thyroid autoantibodies, such as thyroid peroxidase autoantibodies (tpoabstract autoimmune thyroid disease (aitd) is one of the most common endocrinopathies and is more prevalent in women. the circulating thyroid antibodies and the hypothyroidism that often follows aitd have effects on many tissues. the endometrium and ovaries are not spared, and therefore this common morbidity might have an impact on fertility. despite challenging data interpretation and contradictory results, a general takeaway from published studies is that there is a higher incidence of elevated levels of thyroid-stimulating hormone (tsh) and the presence of thyroid antibodies among infertile women. while a single specific and direct pathophysiological mechanism through which autoimmune thyroid disease causes infertility has not been identified, there are multiple gynecological comorbidities that might perpetuate infertility (endometriosis, premature ovarian failure, polycystic ovaries) and defective immunological functions (a shift to a proinflammatory th1 response, increased levels of natural killer cells, cross-reactivity of antigens, etc.) that are affecting fertility. there is insufficient evidence suggesting that levothyroxine (lt4) treatment can help women suffering from aitd conceive and carry out a pregnancy. (kuharić m, rozić d, karner i. thyroid autoimmunity and infertility. seemedj 2017; 1(2); 1-10) seemedj 2017, vol 1, no. 2 thyroid autoimmunity and infertility 2 southeastern european medical journal, 2017; 1(2) ab) and thyroglobulin autoantibodies (tg-ab) induce chronic inflammation in the thyroid gland, which leads to the loss of functional tissue. hypothyroidism can manifest itself with typical signs and symptoms such as fatigue, constipation, depression, thinning hair, cold intolerance, bradycardia, hoarseness, etc. it can also be subclinical (sch), which is classically defined as an increase in thyroid-stimulating hormone (tsh) concentrations over the upper limit of normal range (4.5 – 5.0 miu/l) with normal ft4 levels (5). infertility is defined as an inability to conceive after at least 12 months of continuous intercourse without the use of contraception. its current prevalence among couples ranges between 10 – 15% and has not changed significantly over the past few decades (6 8). because of the effects thyroid hormone has on the ovaries (e.g., maturation of oocytes) and endometrial tissue, it is believed that aitd might play a role in the pathogenesis of infertility. therefore, this review aims to interpret data collected on this subject thus far and summarize published studies to assess the association between aitd and infertility. pathophysiological links between autoimmune thyroid disease and infertility since aitd is the most common endocrine disorder in women of reproductive age, its effects on fertility have been investigated extensively and in detail. nevertheless, a clear pathophysiological link connecting the two morbidities has not been identified and available data suggesting the prevalence of women with both aitd and infertility varies from study to study. furthermore, interpretation of data from published studies on this subject matter is challenging due to several reasons, mostly relating to study designs and their flaws — retrospective studies providing incomplete information, small sample sizes, measurements of different thyroid antibodies, different populations investigated (heterogeneity of ethnicities and geographical locations), etc. variations in sch definitions and tsh ranges additionally contribute to the complexity of data interpretation. the role of thyroid hormones there is a clear connection between the thyroid gland and its hormones and the female reproductive system. thyroid hormone receptors are expressed in the ovary and the endometrium. furthermore, following egg fertilization, the thyroid hormone plays a role in the process of implantation and placentation (9). generally, the proposed pathophysiological mechanisms contributing to infertility in women with aitd are either thyroid-dependent or thyroid-independent. multiple studies have been conducted that aimed to establish the incidence of aitd in infertile women, as well as to connect aitd with certain morbidities affecting the female reproductive system. when observing the data collected and conclusions reached in each of those studies, it can be said that there is a significantly increased incidence of aitd in infertile women compared to controls (10 – 14). in a danish study (10), conducted on 11,254 women, the conclusion reached was that higher tsh levels and higher tpo-ab levels affect fertility. a study by poppe et al. (11) demonstrated significantly higher levels of tsh in infertile subjects compared to controls (1.3 vs 1.1 miu/l), however, tsh levels above or below the normal range were not more prevalent in infertile subjects (with ft4 levels within the normal range), indicating that women suffering from aitd might be affected by fertility issues even though they might seemingly be in a euthyroid state. in addition, women with aitd who are able to conceive are not without risk during pregnancy — a recent study among 10,990 patients, part of the faster (first and second trimester evaluation of risk) trial, shined a light on the connection between thyroid autoimmunity and pregnancy risks. it showed that there was an increased risk for preterm premature rupture of membranes when both tpo-abs and tg-abs were present in both the first and second trimester (15). on the other hand, there have been studies with different results and outcomes. a study by abalovich et al. (16) observed a higher prevalence of sch, but not seemedj 2017, vol 1, no. 2 thyroid autoimmunity and infertility 3 southeastern european medical journal, 2017; 1(2) aitd, in 244 subjects consulting on infertility, providing an example of contradictory data making the interpretation difficult, as mentioned earlier. another example is a study by plowden et al. (17), part of the eager (effects of aspirin in gestation and reproduction) trial. they found that there was no difference in the delay of pregnancy in women with tsh of at least 2.5 miu/l or women with thyroid autoantibodies, compared with those with tsh under 2.5 miu/l or without autoantibodies. couples undergoing art (assisted reproductive technologies) treatments are a group of interest in this context. in a systematic review and metaanalysis of articles describing art outcomes in the context of aitd, busnelli et al. (18) concluded that aitd does not impact the outcome of art treatments (ivf, icsi) in terms of the number of oocytes retrieved and the likelihood of fertilization, implantation and clinical pregnancy. however, a wider implication similar to the one in the faster trial was made, that the presence of thyroid autoantibodies might have harmful effects on the course of the pregnancy, with an increased risk of miscarriage and decreased chance of live birth. poppe et al. also showed that elevated levels of tpo-abs were higher in infertile subjects, while the highest prevalence of positive antibodies among all subgroups of infertility was observed with endometriosis (11). it is a benign condition also linked to altered immune conditions, where endometrial tissue appears outside of the uterus, most commonly in the pelvis, inducing a chronic inflammation (19). endometriosis in the context of aitd has been investigated, but the results have been contradictory. while the study by kim et al. and two other studies (16, 20) reported a higher prevalence of endometriosis in women with aitd versus controls, a study by petta et al. (21) showed that the prevalence of aitd was similar in a group of subjects with endometriosis and in a control group. a more recent study identified anti-laminin-111 autoantibodies as having a potentially major role in the pathogenesis of endometriosisassociated infertility (22). outside of the endometrium, research has been conducted that showed that the source of fertility issues among women with aitd might be the ovary. monteleone et al. (23) hypothesized that the critical source of female infertility due to aitd was in the ovarian follicle itself. their study showed that tg-abs and tpo-abs were measurable in all samples of follicular fluid and serum drawn from subjects with aitd, while they were absent in controls. furthermore, all subjects with aitd were in a euthyroid state, which suggested that the absence of progression of thyroid hormone status towards hypothyroidism due to aitd does not exclude issues with fertility. expanding on that theory, kelkar, et al. (24) demonstrated that human antizona pellucida antigens reacted to murine thyroid tissue, therefore suggesting their similarity in antigens, which suggests, in turn, that zona pellucida may be affected by thyroid autoantibodies. a very common ovarian disorder that may be connected to aitd is polycystic ovary syndrome (pcos). thought to be one of the most common endocrinopathies in women (6.5 – 8 %), it is a complex disease that is multifactorial in its etiology (genetics, obesity, sedentary lifestyle, intrauterine androgen exposure, etc.) it is characterized by hyperandrogenism, increased lh-to-fsh ratio, poor glucose tolerance and hyperinsulinemia and over long-term, it can cause infertility and carries an increased risk for cardiovascular diseases, malignancies, type 2 diabetes mellitus and psychiatric disorders (25). a threefold higher prevalence of aitd in subjects with pcos was demonstrated in a study on 175 subjects, compared to 168 controls, which was, according to the authors of the study, partly correlated with an increased estrogen-to-progesterone ratio (26). further showing that the ovary is a key organ when investigating infertility in women with aitd, in a group of 244 subjects consulting on infertility, aitd prevalence reached statistical significance only in those with premature ovarian failure, which the authors believed to be the result of a shared autoimmune etiology between the two morbidities (16). seemedj 2017, vol 1, no. 2 thyroid autoimmunity and infertility 4 southeastern european medical journal, 2017; 1(2) thyroid hormone independent immunological mechanisms independently of the thyroid, multiple immunological mechanisms have been described as potential contributors to impaired fertility and fecundity in individuals with aitd. a dominant th1 immune response promotes inflammation evoked through cell-mediated mechanisms, which is harmful for pregnancy. th2 cell subsets, on the other hand, regulate and control the inflammation and tissue injury implemented by th1 reactions, as well as protect against autoimmune damage (27). the regulatory mechanisms and balance of th1 and th2 cells are impaired in women with fertility and fecundity issues, with multiple studies confirming a distinct th1 bias (28, 29), even in the endometrial tissue itself (30). a study by kwakkim et al. (30) showed significantly higher th1/th2 ratios of tnf-alfa/il-4 and tnfalfa/il-10 in both women with recurrent pregnancy losses and women with multiple implantation failures, while ifn-gama/il-4, ifngama/il-10 were additionally higher in women with recurrent pregnancy losses. links between impaired t-cell immunity and thyroid autoimmunity have recently been made. tnfalfa/il-10 t-cell ratios were significantly increased in women with aitd (12), thus showing that thyroid antibodies can serve as markers for abnormal immunity, which is an important factor in impaired fertility. when observing abnormal b-cell function and its potential contribution to infertility in women with aitd, a clear connection between the presence of certain non-organ specific antibodies (nosas) and infertile women with aitd has not been proven yet. kim et al. (12) reported that women with aitd did not have a higher prevalence of antiphospholipid antibodies (apas), but did have a higher prevalence of nosas than women with no thyroid antibodies. one study suggested that nosas, in addition to tpo-abs and tg-abs, may serve as independent risk markers for repeated pregnancy loss in women with aitd, but no such statement was made for unexplained infertility (31). aggravation of infertility in women with concurrent thyroid autoimmunity and other systemic autoimmune diseases has also been described, specifically systemic lupus erythematosus (sle) and sjogren's disease (32, 33). in addition to effects t-cells and b-cells might have on the reproductive system, hyperactive and overproduced natural killer (nk) cells can also infiltrate the endometrial tissue and potentially alter the body's immune response, thus affecting fertility (34). tsh has been shown to have a stimulatory effect on nk cells (35) and studies have described increased levels and cytotoxicity of nk cells in aitd (12, 36 38). therefore, nk cells are an additional potential contributor in the pathogenesis of infertility in women with aitd. autoimmune thyroid disease, infertility and nutritional deficiencies yet another contributor linked to both aitd and infertility is vitamin d deficiency. as an omnipresent protein, vitamin d plays a key role in the calcium and phosphate metabolism. it has also been identified as a beneficial factor in many morbidities, including autoimmune diseases, cardiovascular diseases and malignancies (39). vitamin d deficiency (< 10 ng/ml), on the other hand, is, among other things, linked to both aitd and infertility. kivity et al. (40) found that vitamin d deficiency was significantly more prevalent in subjects with aitd than in controls. furthermore, vitamin d deficiency correlated to the presence of thyroid antibodies and abnormal thyroid function. in the context of fertility, vitamin d has been identified as mandatory for reproductive function in the murine model (41). vitamin d deficient rats demonstrate, among other behavioral patterns and physiological changes affecting their reproductive systems, a diminished fertility capacity (42, 43). in humans, the vitamin d receptor (vdr) is expressed in the ovary, the endometrium and even the placenta. furthermore, vitamin d is involved in better ivf outcomes, it plays a role in pcos (deficiency is linked to obesity and metabolic morbidities) and it also affects the regularity of menstrual cycles and influences the production of sex hormones (44). with all that in mind, it is clear how vitamin d deficiency can contribute to both aitd and infertility. seemedj 2017, vol 1, no. 2 thyroid autoimmunity and infertility 5 southeastern european medical journal, 2017; 1(2) there are numerous other microelements and vitamins that play a role in the physiology of thyroid function, so their deficiency, overload or perhaps impaired functionality might also play a role in the development of thyroid autoimmunity and be linked to infertility. these effects haven't been extensively researched, but we know that microelements such as magnesium, iodine, selenium and zinc, as well as other molecules such as riboflavin, vitamin c and coenzyme q10 have been associated with different forms of thyroid disease. magnesium deficiency is the basis of mitochondrial dysfunction which can explain changes associated with thyroid dysfunction (45). an iodine overload increases the risk for an immune reaction, which additionally increases with selenium (se) deficiency (46). se is a necessary trace mineral because of its anti-inflammatory and antioxidant properties. while a study by moncayo et al. (47) did not show a correlation between se deficiency and thyroid autoantibodies, there have been several studies that showed lower se levels in patients with hashimoto thyroiditis (48, 49). se levels were significantly decreased in patients with subacute or silent thyroiditis (47). however, se supplementation during pregnancy and postpartum reduces inflammation in the thyroid and lowers the risk of hypothyroidism (50), while it decreases autoantibody levels and improves the ultrasound structure of the thyroid in patients with hashimoto's thyroiditis (51). antioxidant enzymes also depend on the availability of copper and zinc. a study by stolinska et al. (52) did not confirm, however, that zinc supplementation in patients with normal zinc levels can affect thyroid metabolism. while riboflavin, vitamin c and coenzyme q10 have been researched in the context of thyroid dysfunction, there is insufficient data to show a link between deficiency, overload or dysfunction of those antioxidants with aitd, especially in correlation with infertility (53, 54). hypothyroidism has also been associated with hyperprolactinemia elevated levels of thyrotropin-releasing hormone (trh) and tsh cause increased secretion of prolactin. studies have shown that women of fertile age are most commonly affected by hyperprolactinemia as well as its presence in sch, showing yet another consequence of this silent morbidity (55, 56). it can also impair fertility, since elevated prolactine levels and the pulsatile gnrh secretion may lead to a delayed lh response and affect corpus luteum function (56 – 58). finally, thyroid antibodies could also inhibit the activity of human chorionic gonadotropin (hcg) on the corpus luteum due to cross-reactivity that has been observed between hcg and tsh. the corpus luteum plays a key role in supporting and maintaining a pregnancy in the first trimester through progesterone and estrogen secretion and is largely dependable on hcg. this pathophysiological mechanism doesn't cause infertility per se, but it may impair fecundity, since tsh receptor blocking antibodies could also block luteinizing hormone (lh) and hcg receptors, causing a decrease in steroid hormone production, resulting in spontaneous miscarriages (59). the american thyroid association published guidelines for the diagnosis and management of thyroid disease during pregnancy and the postpartum in 2017. they stated that evaluation of serum tsh concentration is recommended for all women seeking care for infertility. additionally, they recommended levothyroxine (lt4) treatment for infertile women with overt hypothyroidism who desire pregnancy. however, no such recommendation was made for women suffering from aitd and euthyroid women due to insufficient evidence to determine whether or not lt4 therapy actually improves fertility in such cases. the only case where administration of low-dose lt4 was to be considered was in infertile women suffering from sch, but were thyroid antibody-negative (60). there are several examples of conflicting data and evidence that is insufficiently strong to suggest lt4 therapy improves the chances of achieving pregnancy. negro et al. (61) investigated the effects of lt4 treatment in tpoab positive women undergoing assisted reproduction technologies (art) and found that the pregnancy rate was not affected by treatment with lt4. in the study by abalovich et seemedj 2017, vol 1, no. 2 thyroid autoimmunity and infertility 6 southeastern european medical journal, 2017; 1(2) al (16), however, lt4 was prescribed to 34 subjects with sch and after 6 months of followup, 44.1% achieved pregnancy. conclusion aitd and infertility are two morbidities that have separately been widely investigated and detailed pathophysiological mechanisms for both entities have been described. however, there is a wide variety of overlapping factors that make it difficult to distinguish how and if aitd and infertility are interconnected. all the factors described in this review (abnormal lymphocyte production, a th1 immune response bias, vitamin d deficiency, etc.) (figure 1.) could be contributing to infertility as well as aitd to some extent, but the possibility remains that one or a few of them might have a more significant impact on the pathogenesis. conflicting data and different study designs make shedding light on the interconnectedness of these morbidities difficult. there is even no consensus on the exact organ of origin of infertility caused by thyroid autoimmunity, with the uterus and the ovary both potentially playing roles and being targeted by the thyroid antibodies. based on the findings of the studies, there is still an insufficient amount of evidence that would suggest levothyroxine treatment is a viable treatment option for infertile women suffering from aitd. recommendations from 2015 have only been made for infertile women suffering from sch, but who are thyroid antibodynegative. even in those cases, however, lt4 treatment should simply be "considered". that is figure 1. clinical entities and pathophysiological mechanisms linking female infertility with aitd. (tsh – thyroid-stimulating hormone; sle – systemic lupus erythematosus; nosas – non-organ specific antibodies; hcg – human chorionic gonadotropin; nk – natural killer; pcos – polycystic ovary syndrome; pof – premature ovarian failure) seemedj 2017, vol 1, no. 2 thyroid autoimmunity and infertility 7 southeastern european medical journal, 2017; 1(2) why it is crucial to continue researching these two entities to potentially increase the chances for women to conceive and carry out a healthy pregnancy. disclosures funding: no specific funding was received for this review article. there is no conflict of interest to be declared. references 1. yen pm. physiological and molecular basis of thyroid hormone action. physiol rev 2001;81(3):1097-142. 2. song y, yao x, ying h. thyroid hormone action in metabolic regulation. protein cell 2011;2(5):358-68. 3. hollowell jg, staehling nw, flanders wd, hannon wh, gunter ew, spencer ca, et al. serum tsh, t(4), and thyroid antibodies in the united states population (1988 to 1994): national health and nutrition examination survey (nhanes iii). j clin endocrinol 2002;87(2):489-99. 4. bjoro t, holmen j, kruger o, midthjell k, hunstad k, schreiner t, et al. prevalence of thyroid disease, thyroid dysfunction and thyroid peroxidase antibodies in a large, unselected population. the health study of nord-trondelag (hunt). eu j endocrinol 2000;143(5):639-47. 5. fatourechi v, klee gg, grebe sk, bahn rs, brennan md, hay id, et al. effects of reducing the upper limit of normal tsh values. jama. 2003;290(24):3195-6. 6. mosher wd, pratt wf. fecundity and infertility in the united states: incidence and trends. fertil steril. 1991;56(2):192-3. 7. mascarenhas mn, flaxman sr, boerma t, vanderpoel s, stevens ga. national, regional, and global trends in infertility prevalence since 1990: a systematic analysis of 277 health surveys. plos medicine. 2012;9(12):e1001356. 8. chandra a, copen ce, stephen eh. infertility and impaired fecundity in the united states, 1982-2010: data from the national survey of family growth. national health statistics reports. 2013(67):1-18, 1 p following 9. 9. vissenberg r, manders vd, mastenbroek s, fliers e, afink gb, ris-stalpers c, et al. pathophysiological aspects of thyroid hormone disorders/thyroid peroxidase autoantibodies and reproduction. hum reprod update 2015;21(3):378-87. 10. feldthusen ad, pedersen pl, larsen j, toft kristensen t, ellervik c, kvetny j. impaired fertility associated with subclinical hypothyroidism and thyroid autoimmunity: he danish general suburban population study. j pregnan 2015;2015:132718. 11. poppe k, glinoer d, van steirteghem a, tournaye h, devroey p, schiettecatte j, et al. thyroid dysfunction and autoimmunity in infertile women. thyroid2002;12(11):9971001. 12. kim ny, cho hj, kim hy, yang km, ahn hk, thornton s, et al. thyroid autoimmunity and its association with cellular and humoral immunity in women with reproductive failures. am j reprod immunol 2011;65(1):7887. 13. kutteh wh, yetman dl, carr ac, beck la, scott rt, jr. increased prevalence of antithyroid antibodies identified in women with recurrent pregnancy loss but not in women undergoing assisted reproduction. fertil steril 1999;71(5):843-8. 14. bellver j, soares sr, alvarez c, munoz e, ramirez a, rubio c, et al. the role of thrombophilia and thyroid autoimmunity in unexplained infertility, implantation failure and recurrent spontaneous abortion. hum reprod 2008;23(2):278-84. 15. cleary-goldman j, malone fd, lambertmesserlian g, sullivan l, canick j, porter tf, et al. maternal thyroid hypofunction and pregnancy outcome. obstetrics and gynecology. 2008;112(1):85-92. seemedj 2017, vol 1, no. 2 thyroid autoimmunity and infertility 8 southeastern european medical journal, 2017; 1(2) 16. abalovich m, mitelberg l, allami c, gutierrez s, alcaraz g, otero p, et al. subclinical hypothyroidism and thyroid autoimmunity in women with infertility. gynecol endocrinol 2007;23(5):279-83. 17. plowden tc, schisterman ef, sjaarda la, zarek sm, perkins nj, silver r, et al. subclinical hypothyroidism and thyroid autoimmunity are not associated with fecundity, pregnancy loss, or live birth. j clin endocrinol 2016;101(6):2358-65. 18. busnelli a, paffoni a, fedele l, somigliana e. the impact of thyroid autoimmunity on ivf/icsi outcome: a systematic review and meta-analysis. hum reprod update2016;22(6):775-90. 19. matarese g, de placido g, nikas y, alviggi c. pathogenesis of endometriosis: natural immunity dysfunction or autoimmune disease? 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7(1) review article home-based exercise during the coronavirus pandemic – a useful, yet challenging treatment strategy for improvement of mental health, glycemic control and covid-19 outcomes in patients with diabetes 1 maja cigrovski berković 1,2, marul ivandić 3, anna mrzljak 4,5, vjekoslav cigrovski 2, lavinia la grasta sabolić 6, klara ormanac 7, dea sabo 7, tea omanović kolarić 7,8, lana ružić 2, ines bilic-ćurčić 3,7* 1 department of endocrinology, diabetes, metabolism and clinical pharmacology, clinical hospital dubrava, zagreb, croatia 2 department of kinesiological anthropology and methodology, faculty of kinesiology, university of zagreb, zagreb, croatia 3 university hospital center osijek, osijek, croatia 4 department of gastroenterology and hepatology, university hospital center zagreb, zagreb, croatia 5 school of medicine, university of zagreb, zagreb, croatia 6 department of pediatric endocrinology and diabetes, university hospital center sestre milosrdnice, zagreb, croatia 7 department of pharmacology, faculty of medicine, university of j. j. strossmayer osijek, osijek, croatia 8 department of pharmacology and biochemistry, faculty of dental medicine and health, university of j. j. strossmayer osijek, osijek, croatia *corresponding author: ines bilić ćurčić, ibcurcic@mefos.hr received: mar 7, 2023; revised version accepted: apr 27, 2023; published: apr 30, 2023 keywords: physical activity, mental health, diabetes, covid-19, home-based exercise abstract lockdown measures to control disease transmission were implemented at the start of the covid-19 era, worsening the already existing sedentary lifestyle. reduced physical activity (pa) and unhealthy eating habits have a negative impact on mental health in chronically ill patients, including diabetes patients. mental illness, on the other hand, encourages a sedentary lifestyle, exacerbating all components of metabolic syndrome. while well-controlled diabetic patients with an hba1c of less than 7% had a less severe clinical presentation and covid-19 mortality rates, the favorable effect of pa on immunomodulation and immunoregulation should not be neglected. given recent data indicating that a sedentary lifestyle is the third independent risk factor for covid-19 complications and death (after advanced age and organ transplant), including regular pa has never been more vital. since pa has a major impact on both glycemic control and mental health, implementing structured home-based activity programs could improve glycemic control and psychological well-being, hence positively impacting covid-19 outcomes. (cigrovski berković m, ivandić m, mrzljak a, cigrovski v, la grasta sabolić v, ormanac k, sabo d, omanović kolarić t, ružić l, bilic-ćurčić i. home-based exercise during the coronavirus pandemic – a useful, yet challenging treatment strategy for improvement of mental health, glycemic control and covid-19 outcomes in patients with diabetes. seemedj 2023; 7(1); 28-35) seemedj 2023, vol 7, no 1 home-based exercise during the coronavirus pandemic 29 southeastern european medical journal, 2023; 7(1) introduction physical activity is defined as any movement produced by skeletal muscles that require energy expenditure (1). it refers to all activities performed while traveling, during leisure time or working hours. physical activity should be viewed as a continuum, ranging from light to vigorous, while exercise implies planned, structured and repetitive physical activity. overall, it is crucial for the improvement and/or maintenance of both mental and physical health (2, 3). different psychological and physiological mechanisms have been proposed to explain the positive effects of physical activity on mental health, especially on anxiety, depression and stress-related disorders (4, 5, 6). potential psychological mechanisms include “time out” or distraction from stressors, as well as improved self-efficacy, self-image and confidence, which enhance the sense of control and help to overcome the difficulties faced on a daily basis (7). from the physiological aspect, exercise may induce changes in the hypothalamic-pituitaryadrenal axis modulating stress reactivity (4). it may also boost the levels of endorphins, which help people feel happier and less stressed (8). besides, the brain-derived neurotrophic factor increases with exercise, thus contributing to the anxiolytic effect of physical activity (9). it is well known that physical inactivity and sedentary behavior represent the key modifiable factors associated with an increased risk of developing type 2 diabetes (t2d). nowadays, diabetes is the fastest-growing chronic disease strongly related to rising obesity rates. regular physical activity constitutes an important part of diabetes/obesity prevention and management. it produces multiple health benefits including enhanced glycemic control, favorable changes in blood lipid levels, reduced low-grade inflammation and improved vascular function (10, 11). physical activity and exercise also reduce all-cause and diabetes-related mortality in t2d. mechanisms enabling those benefits are complex and mediated by an increase in glycolipid uptake and utilization, improved insulin sensitivity, optimized body mass index and modulated dna methylation (12). recent findings also suggest that exercise induces significant physiological changes in the immune system, including anti-inflammatory cytokine response (12, 13). for people with type 1 diabetes (t1d), physical activity improves cardiovascular fitness, muscle strength and insulin sensitivity (14, 15). many patients suffering from chronic diseases have co-morbid mental health conditions (16). diabetes and mental health issues share a bidirectional association influencing one another in multiple ways. in patients with diabetes, the prevalence of anxiety and depression is significantly higher than in the general population (17, 18). depression in combination with behavioral and metabolic risk factors increases the risk for type 2 diabetes development, poor glycaemic control and subsequent risk for micro and macrovascular complications. it is important to emphasize that being diagnosed with diabetes represents a strong stressor by itself, requiring a large number of physical and mental adjustments. the adoption and maintenance of regular physical activity are beneficial for both, diabetes and mental health disorders. however, many patients neither meet their exercise targets, nor adhere to proposed exercise guidelines. between 25% and 42% of older diabetics meet the physical activity recommendations, while 13–19% are not even likely to be physically active at sufficient levels compared to non-diabetic persons (19). additionally, more than 60% of patients with t1d remain sedentary (20). people with severe mental illness engage in significantly less vigorous exercise and express greater amounts of sedentary behavior in comparison with healthy controls (21). their inactivity is predictive of a range of adverse health outcomes including metabolic syndrome and its components (22). it is reasonable to assume that number of physically inactive people with diabetes and mental health disorders increased during the coronavirus pandemic. knowledge of commonly perceived barriers to physical seemedj 2023, vol 7, no 1 home-based exercise during the coronavirus pandemic 30 southeastern european medical journal, 2023; 7(1) activity, along with psychological factors contributing to these barriers may help in overcoming challenges of sedentarism both in non-pandemic and pandemic covid-19 times. the challenge of physical activity in non-pandemic times according to current guidelines, people with diabetes should engage in five sessions of moderate aerobic activity weekly (at least 150 min/weekly, i.e. five days per week with no more than two consecutive days between the activity) to maintain the exercise-induced improvements in insulin action. besides aerobics, resistance training is recommended three times weekly on nonconsecutive days (23). transcribed into parameters of glucoregulation, a decrease in hba1c by up to 0.85% can be expected, which matches the reductions seen with some newer antidiabetic agents. so, the intriguing question is why physicians do not prescribe physical activity more persuasively, and why are patients so frequently noncompliant and nonadherent? the assumptions for successful prescription of physical activity imply that medical professionals know how to prescribe, monitor and evaluate the effectiveness of the exercise prescribed (adapting exercise type, frequency, intensity and duration according to patients’ individual health status and interests) which is generally not the case. other known obstacles to physical activity promotion include lack of time and lack of perceived efficacy in changing physical activity behavior in patients (24). the most typical barriers perceived by t2d patients are lack of time, pain and discomfort, being overweight and finding physical activity boring (25). interestingly, a commonly reported reason for avoiding exercise among overweight t2d females is embarrassment about their appearance. in contrast, in t1d patients, fear of hypoglycemia pops up as the most prevalent reason behind not exercising (26). patients with diabetes are sedentary, spending too much time viewing tv, which represents an independent risk factor for the increase of all-cause mortality (27). in the case of co-morbid mental health issues, mental health symptoms and tiredness may further complicate exercise performance. for people with severe mental disorders, additional factors that hamper physical activity include medication side effects, complications from obesity and poor physical health, lack of resources and absence of professional support (28). covid-19 pandemic and restrictions related to physical activity the covid-19 pandemic imposes challenges for people with diabetes and mental health disorders, tackling their health and well-being. lockdowns and mobility restrictions impacted the ability to be active during work and leisure time. many people worked through home-based offices, leaving little or no time for movement and increasing screen time, sedentarism and unhealthy meal plans (29). moreover, pandemicdriven economic and financial distress distracted people from engaging in physical activity (30) and weakened their motivation to exercise (31). studies reported direct negative effects on glucoregulation of t1d patients during lockdown (32). furthermore, high physical inactivity found in most t2d patients before the covid-19 pandemic additionally worsened during the lockdown (33). at the same time, public health measures such as quarantine, selfisolation and social distancing contributed to the deterioration of mental health and increased stress, anxiety, depression and feelings of isolation (34, 35). in light of the coronavirus pandemic, the beneficial effects of physical activity on immune function and inflammation can be especially important for people with t2d and mental health issues, as they both have a higher risk of covid19 hospitalization and death (36, 37). also, the favorable impact of physical activity on glycemic control is not negligible because it may help reduce the chance of severe covid-19 in diabetic patients (38). it is important to point out that only advanced age and a history of organ transplant are stronger independent risk factors for severe covid-19 outcomes than physical inactivity (39). on the contrary, engaging in various types of exercise is considered to be a coping strategy for mitigating the negative seemedj 2023, vol 7, no 1 home-based exercise during the coronavirus pandemic 31 southeastern european medical journal, 2023; 7(1) effects of the coronavirus pandemic on mental health and enhancing general well-being (40). home-based exercise – could it make a difference? as a part of the covid-19 social distancing measures, the use of gyms, health clubs and public spaces was either reduced, not recommended, or not permitted, especially for the high-risk populations. the restricted collective activities during the pandemic imposed the importance of physical activity through alternative forms of exercise, especially home-based exercise (hbe). therefore, knowing the benefits of regular physical activity and exercise on glycemic management and mental health, hbe training emerges as an important approach and coping strategy to promote physical and psychological well-being during the pandemic times. several randomized control trials, including people at risk for contracting t2d, patients with t2d and different cardiovascular profiles and women with gestational diabetes mellitus showed health advantages of hbe (primarily moderate-intensity aerobic exercise 3–5 times weekly and less often combined aerobic and resistance exercise), during the time of intervention and follow-up (table 1). benefits were, besides glycemic control, seen in lipid profile, body composition, cardiorespiratory fitness and psychological health (41). patients adherent to the hbe program had a significantly reduced incidence of cardiovascular disease compared to nonadherent patients (42). in addition, none of the studies reported adverse events during the hbe programs or follow-up. studies provided material about the exercise program, education on diabetes selfmanagement, heart rate monitors and oximeters which helped the participants in safe exercising (43, 44). unfortunately, no studies assessed the safety and effectiveness of hbe programs in t1d individuals, but with modern technologies such as continuous and flash glucose monitoring and the use of insulin pumps, one can speculate their usefulness and safety during covid-19 (45). table 1. selected findings from different studies regarding the effectiveness of various exercise programs author title source findings francesco cosentino 2019 esc guidelines on diabetes, pre-diabetes and cardiovascular diseases developed in collaboration with the easd european heart journal clinical trials in adults with dm have provided evidence of the hba1c-lowering value of resistance training, and of an additive benefit of combined aerobic and resistance exercise. s. f. lee an investigation and comparison of the effectiveness of different exercise programs in improving glucose metabolism and pancreatic β cell function of type 2 diabetes patients the international journal of clinical practice the accumulated million steps group had better glucose metabolism and pancreatic β cell function compared with those in the aerobic exercise group. jos j kraal clinical and costeffectiveness of home-based cardiac rehabilitation compared to conventional, center-based cardiac rehabilitation: results of the fit@home study european journal of preventive cardiology patients in the home-based group were more satisfied with their cr program compared to patients in the center-based group (homebased: 8.7/10, center-based: 8.1/10, p = 0.02). seemedj 2023, vol 7, no 1 home-based exercise during the coronavirus pandemic 32 southeastern european medical journal, 2023; 7(1) the italian national association of athletes with diabetes (aniad) issued a series of daily activities that could be performed home-based, and with an intensity comparable to a one-hour brisk walking with an energy expenditure of 150– 200 kcal (46). the above-mentioned could not only secure the reaching and attainment of recommended physical activity guidelines for people with diabetes, but also enhance access to exercise and its adjustment to patients’ lifestyles, daily schedules, and integration with regular home routines, all of which are often depicted by patients as barriers and obstacles for participation in regular physical activity. hbe does not always have to be high-tech and designed on platforms aimed exclusively at diabetes patients. those narrowly specialized platforms would be best, but their reach could be limited, resulting in a low number of users. the solution might be in general platforms, which give the possibility to choose the location and type of activity. the newest addition is online programs. as the infrastructure already exists, there is a possibility to offer online programs explicitly created for diabetes patients. the collaboration between diabetes centers and such platforms would speed up the online exercise offer by using existing software solutions and a large pool of members. nearly all types of physical activity are helpful since both physical exercise and relaxation training can buffer the negative effects of stress on mental health (47). hbe can include aerobic activities such as dancing (35), balance and flexibility training, such as yoga (48), muscle strengthening exercises, such as weightlifting (35), endurance training and others. participation in physical activities at home has been shown to alleviate a wide range of mental illness symptoms, improve anxiety, mood, social and emotional health (34, 49). studies have shown that yoga, as an example of indoor physical activity, can reduce perceived stress, enhance emotional control and improve self-efficacy, self-confidence and overall quality of life (50). it can be used alone or in combination with other interventions (48), and it is one of the important measures to prevent or control mental health problems during the pandemic. exergames, which are exercises based on video games, can encourage younger people to exercise at home, while the internet may further enable social interaction with friends remotely (51). conclusions physical inactivity emerges as an important issue with deleterious physical and mental health consequences. in patients with diabetes, regardless of the disease type, regular exercise has well-known and proven benefits on glycemic control and cardiovascular health. moreover, physical activity can reduce psychological pressure, promote mental health and improve the quality of life. in the context of the covid-19 pandemic, maintaining regular exercise is especially beneficial, and engaging in alternative modes of physical activity such as hbe can be a key strategy to maintain mental health and physical well-being. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. world health organization. physical activity. 2018; 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21:100408 48. ransing r, da costa mp, adiukwu f, grandinetti p, teixeira als, kilic o, soler-vidal j, ramalho r. yoga for covid-19 and natural disaster related mental health issues: challenges and perspectives. asian j. psychiatr 2020; 53:102386 49. puyat jh, ahmad h, avina-galindo am, kazanjian a, gupta a, ellis u, ashe mc, vila-rodriguez f, halli p, salmon a, vigo d, almeida a, de bono ce. a rapid review of home-based activities that can promote mental wellness during the covid-19 pandemic. plos one 2020; 15:e0243125 50. wang f, szabo a. effects of yoga on stress among healthy adults: a systematic review. alternative ther. health med 2020; 26:at6214 51. viana rb, de lira cab. exergames as coping strategies for anxiety disorders during the covid-19 quarantine period. games health j 2020; 9:147–9. author contribution. acquisition of data: mcb, mi, am, vc, lgs, ko, ds, tok, lr, ibc administrative, technical or logistic support: mcb, mi, am, vc, lgs, ko, ds, tok, lr, ibc analysis and interpretation of data: mcb, mi, am, vc, lgs, ko, ds, tok, lr, ibc conception and design: mcb, mi, am, vc, lgs, ko, ds, tok, lr, ibc critical revision of the article for important intellectual content: mcb, mi, am, vc, lgs, ko, ds, tok, lr, ibc drafting of the article: mcb, mi, am, vc, lgs, ko, ds, tok, lr, ibc final approval of the article: mcb, mi, am, vc, lgs, ko, ds, tok, lr, ibc guarantor of the study: mcb, mi, am, vc, lgs, ko, ds, tok, lr, ibc obtaining funding: mcb, mi, am, vc, lgs, ko, ds, tok, lr, ibc provision of study materials or patients: mcb, mi, am, vc, lgs, ko, ds, tok, lr, ibc seemedj 2022, vol 6, no 2 iron chelation therapy in covid-19 infection 75 southeastern european medical journal, 2022; 6(2) review article iron chelation therapy in covid-19 infection: a review article 1 irena krajina kmoniček 1, anja tomić 2, josip kocur 3 1 department of anesthesiology, reanimatology and intensive medicine, osijek university hospital centre, osijek, croatia 2 community health centre vinkovci, vinkovci, croatia 3 department of orthopedics and traumatology, osijek university hospital centre, osijek, croatia *corresponding author: irena krajina kmoniček, ikrajina20@gmail.com received: mar 19, 2022; revised version accepted: oct 28, 2022; published: nov 28, 2022 keywords: covid-19, sars-cov-2, iron chelating agents, covid-19 therapy abstract the recent outbreak of corona virus and coronavirus disease (covid-19) caused by the sars-cov-2 virus is a global concern. despite efforts to clarify the physiology and potential therapy, specific guidelines for the treatment of covid-19 disease have yet to be established, and many therapeutic options are under investigation. accumulating evidence suggests that dysregulation of iron homeostasis contributes significantly to the pathogenesis of covid-19 through its toxic effects by the formation of reactive oxygen species (ros). this review focuses on summarizing the available literature and relevant studies conducted to date on the possible therapeutic effects of iron chelation therapy in the treatment of covid-19 disease. scientific databases (pubmed, scopus, google scholar) were searched for relevant articles using the following keywords: covid-19, sars-cov-2, coronavirus, clinical management, iron chelators/chelation. research articles, reviews, research letters, case reports, and commentaries were considered. although there is ample evidence of the potential beneficial effects of using iron chelators as adjuvant treatment in covid-19, further research on this topic is needed. (krajina kmoniček i, tomić a, kocur j. iron chelation therapy in covid-19 infection: a review article. seemedj 2022; 6(2); 75-84) seemedj 2022, vol 6, no 2 iron chelation therapy in covid-19 infection 76 southeastern european medical journal, 2022; 6(2) the novel disease in december 2019, a new virus from the coronavirus group (initially 2019-ncov) emerged and caused the appearance of unusual viral pneumonia in china. the disease is called coronavirus disease (covid-19) and in march 2020, the world health organization declared covid-19 a pandemic. the 2019ncov was later named sars-cov-2 due to its structural similarity to the sars-cov virus that caused the 2003 sars outbreak (1). vaccination campaigns against the sars-cov-2 virus are currently underway worldwide, but the covid-19 pandemic is still out of control and continues to cause high mortality. unfortunately, there is still no specific therapy for covid -19 and patients rely on general and supportive therapies. although drugs (antiviral drugs, monoclonal antibodies, corticosteroids, immunosuppressants) included in the recommended guidelines for the treatment of the infection show promising results, given the rising number of covid-19 cases, additional therapeutic choices should be identified and thoroughly evaluated (2-4). the goal of therapy is to prevent the occurrence of a cytokine storm and to avoid significant damage to body tissues resulting in multiorgan failure and death. most cases have a milder clinical course, but up to 14% of cases can be severe, and present with moderate to severe pneumonia requiring hospitalization. severe cases are characterized by dyspnea, tachypnea (rr ≥ 30/min), hypoxemia (spo2 ≤ 93%), pao2/fio2 ratio < 300, and/or pulmonary infiltrates involving more than 50% of the lung parenchyma. all of this can lead to severe disease requiring intensive care unit (icu) treatment and can be life-threatening in 5% of cases characterized by respiratory failure that can provoke the development of acute distress syndrome (ards), septic shock, multiple organ dysfunction and death (5). risk factors for fatal outcome include age, underlying comorbidities (hypertension, diabetes mellitus, obesity, heart failure, coronary artery disease), and disease severity, which increases by up to 49% in critically ill patients (6,7). in addition, coronavirus also affects numerous other organ systems, such as the cardiovascular system (e.g., myocardial damage, cardiomyopathy, and cardiac arrhythmia (8-11), the neurological system, and can also cause acute kidney injury and liver damage (12,13). inflammation-induced coagulopathy, which causes an elevated coagulation state, is a consequence of damage to the endothelium and the action of pro-inflammatory cytokines (especially il-6) (14-16). endothelial cells are potential targets for sars-cov-2 due to the highly expressed ace 2 receptors, which are thought to be the major (but not the only) port of entry into a cell for the virus (17). the virus also recognizes porphyrin in hemoglobin, with higher binding affinity than that to hace2, resulting in oxygen deprivation (17). in addition to endothelial cells and porphyrin, transferrin receptors (tfr) are also considered as a possible target of viral action(18, 19). tfr is found on numerous tissues and cells, including cells of the respiratory system. transferrin, a circulating glycoprotein that transports iron, delivers iron to cells when it binds to tfr. studies in animal models have shown that viral infection did not occur when connection between virus and tfr was affected (18). iron and ferritin in inflammation iron metabolism is very important for the functioning of the whole body and ferritin plays a crucial role in this process. ferritin is a protein that binds iron and is found in the bloodstream, cytosol and mitochondria. it is involved in crucial cellular activities, including immune regulation by making iron available and protecting cells from the toxic performance of free iron (20). by measuring serum ferritin levels, one can get an insight into iron status. during inflammation, there is often an increase in serum ferritin concentration with hypoferremia. oral iron supplementation drugs have been shown to increase mortality in humans when taken during infection (20). serum ferritin synthesis, apart from iron availability, is also regulated by inflammatory cytokines such as il-1β and il-6 (pro-inflammatory cytokine in covid-19 infection) and increased hepcidin production, which in turn is stimulated by pro-inflammatory cytokines, especially il-6 (21). to survive and seemedj 2022, vol 6, no 2 iron chelation therapy in covid-19 infection 77 southeastern european medical journal, 2022; 6(2) replicate in host cells, microbes require iron and to limit viral replication, the innate immune system takes control of iron metabolism by reducing iron bioavailability (2). to protect the host during active infection, ferritin reduces iron bioavailability to the pathogen. as a result, serum iron concentration decreases and serum ferritin concentration increases, limiting the availability of iron for erythropoiesis and leading to further exacerbation of anemia, which is called anemia of inflammation (ai) (22). ferritin also plays a role in inflecting the immune response by inducing anti-inflammatory cytokines and limiting free radical-induced damage (23). inflammation and infection produce large amounts of oxygen radicals (ros) that leak into the fluids and tissues in the area of inflammation, causing cellular damage that can lead to endothelial dysregulation of the immune response, resulting in hyperinflammation and cytokine storms and multiple organ failure (24). when the concentration of non-transferrin bound iron in plasma is too high, it converts to its redox-active form called labile plasma iron. this further contributes to the production of ros, leading to tissue lesion and, over time, fibrosis (5). these toxic free radicals are formed by the fenton reaction, in which, in the presence of a harmful byproduct of aerobic metabolism hydrogen peroxide, ferrous iron (fe2+) is oxidized to ferric iron (fe3+), producing a hydroxyl radical and hydroxide ion. in addition to ferritin, transferrin and its effect on iron must also be mentioned, as it has been shown that the concentration of transferrin changes during covid-19 disease. transferrin is a glycoprotein synthesized by the liver. it is found in the bloodstream, where it transports iron to tfr receptors on cells. when there is iron deficiency in the body and hypoxia, transferrin concentration increases, while it decreases during inflammation (18). transferrin saturation with iron (tsat) indicates how much iron is bound to transferrin and is an important marker for iron availability and the amount of systemic iron (18). the role of iron in covid-19 infection dysregulation of iron homeostasis, including iron overload, has also recently been recognized as an important element in the pathogenesis of covid-19, along with high levels of proinflammatory cd4 and cd8 t cells, extensive cytokine release, and an increased coagulation state. as mentioned earlier, elevated ferritin levels not only indicate an acute phase response, but also play an important role in inflammation by contributing to the progression of cytokine storm (20). during cytokine storm there is an enormous and uncontrolled release of pro-inflammatory cytokines (il-6, il-10, tnfα, il-1β, ifn-γ, il-2, il-7 and il-10, g-csf, mip-1 alpha, and others), which is especially prominent in more severe clinical forms of covid-19 disease. as a result of the cytokine storm and the damaging cytopathic effect of the virus, there is destruction of the lungs and other organs and, at the same time, a further increase in cytokine levels. besides the cytokine storm, high levels of intracellular iron generate ros interaction with oxygen molecules and increases the risk of coagulopathy, oxidative stress and endothelial inflammation, all of which together can lead to disseminated coagulopathy and multiorgan failure (25). according to current clinical and experimental data, it is possible that significant oxidative stress may cause the progression of ards characterized by damage to the lung parenchyma, decreased lung capacity, endothelial and capillary membrane damage resulting in protein leakage (24). during this tissue damage and lysis, there is an additional increase in the level of ferritin, the synthesis of which is already elevated due to ongoing inflammation (26). moreover, analysis of lavage fluid from patients with ards shows increased levels of iron and increased cellular levels of transferrin, ferritin, and lactoferrin, implying interruption of pulmonary iron homeostasis in ards (4). as mentioned earlier, during covid-19 infection, there is an iron overload and little attention is paid to this finding. several studies suggest that patients with high ferritin levels have a much more severe form of covid-19 disease, clinical deterioration of the patient's condition, a higher seemedj 2022, vol 6, no 2 iron chelation therapy in covid-19 infection 78 southeastern european medical journal, 2022; 6(2) mortality rate, and worse outcome in patients treated in the icu (23,24). hyperferritinemia is linked with considerably elevated mortality in septic patients, which has also been shown in patients with severe covid-19 infection in icu% (5). patients with covid-19 disease and ferritin levels above 300 μg/l had a 9-fold higher risk of death4. although little is well-known about the management of iron balance in sars-cov-2 patients, some conclusions can be drawn from other viral infections such as hepatitis b, hepatitis c, and hiv, in which iron overload leads to a worse prognosis (5,27). in hiv infection, for example, hiv 1 replication is dependent on host cell enzymes that require iron, and iron supplementation has been shown to lead to increased mortality in hiv-infected patients, indicating the importance of iron excess in hiv infection (2). the level of transferrin and tsat, mentioned earlier in the text, proved useful in assessing the severity of covid-19 disease and survival. in patients hospitalized for covid-19 disease, the serum concentration of transferrin decreases, although at the same time a low serum iron level is present (19, 28-31). it is possible that covid-19 inflammation regulates the action of transferrin and prevents its increase when low iron levels are present (19). a very low concentration of transferrin was observed in patients who required oxygen therapy, and a continuous decrease in concentration was observed in patients who died (19, 28-31). in patients who survive, serum transferrin levels recover after some time. the tsat decreases in covid-19 patients, especially in patients who have a severe form of the disease and are in the icu (19, 30). the drop in tsat level is explained by the fact that at the beginning of the infection, the availability of iron for the pathogen is limited. after a few days, the tsat level recovers and returns to normal range. it is interesting to note that tsat levels were higher in intubated patients than in non-intubated patients, which may be explained by changes in the regulation of metabolism at different stages of the disease or by the effect of the tube on iron metabolism (19). overexpression of il-6, il-1β, and ifn-γ during inflammation also leads to an increase in hepcidin levels (5). hepcidin is an iron-regulating peptide hormone produced in the liver and released into the bloodstream in response to inflammation and increased iron levels in the body. the production of hepcidin in the liver is stimulated by il-6 (32). it is a negative regulator of iron by sequestering iron in enterocytes and macrophages, increasing intracellular ferritin levels, and preventing iron efflux from storage cells by inhibiting ferroportin (33). it is possible that sars-cov-2 virus has a hepcidin-like effect because of the identical amino acid sequence between hepcidin and the coronavirus spike glycoprotein. by mimicking the action of hepcidin, sarscov-2 could remarkably increase circulating and tissue ferritin (especially in liver, spleen, bone marrow, and muscle) independent of inflammation, while causing serum iron deficiency and hemoglobin deficiency (4, 32). it is also possible that coronaviruses enter cells through complex mechanism by a mimic effect using their spike proteins and cleave their spike polypeptides using host furins and proteases, which promotes cell entry (32). covid-19 infection resembles hyperferritinemic syndromes due to high blood ferritin levels and inflammation triggered by the cytokine storm, as well as lymphopenia, decreased nk count and activity, abnormal liver function tests, coagulopathy, pleurisy, pericarditis, lung consolidation, pulmonary edema, and myocarditis (34,35). because iron chelation is the basis for treating iron overload, as it is in other hyperferritinemic syndromes, and because impaired iron metabolism has been observed in covid-19 infection, iron chelator therapy may be beneficial. there are several theories about how increases in ferritin and free iron may occur during covid19 infection. an in-silico model suggests and considers direct interaction between several viral proteins and hemoglobin, but side effects on inflammation or tissue damage are not considered (36, 37). the viral proteins (orf1ab, orf10, orf3a) originate from infected plasma cells and together remove heme from the bseemedj 2022, vol 6, no 2 iron chelation therapy in covid-19 infection 79 southeastern european medical journal, 2022; 6(2) chain of hemoglobin, remove iron from heme, and consequently sequester iron-free protoporphyrin ix (ppix). as a result, a toxic amount of iron is released, functional hemoglobin levels are impaired and hemoglobin metabolism is disturbed. another theory, as addressed previously in the text, is that ai may be the cause of the decreased hemoglobin level (2, 22). iron chelator therapy iron chelators have several beneficial properties such as chelating iron, inhibiting the redox properties of free iron, and preventing the involvement of iron in fenton reactions. they inhibit the production of hydroxyl radicals and the production of other ros which lead to oxidative damage and ferroptosis (38). another useful mechanism of iron chelators is the downregulation of hepcidin and the removal of iron from iron-binding proteins, showing their anti-ferritin effect (2,39). fda-approved iron chelators such as deferoxamine (dfo), deferiprone, and deferasirox have so far been used as iron overload therapy in a number of pathogens in vivo and in vitro, particularly (dfo) (17). each of the iron chelators has different efficacy in iron overload therapy. dfo could be effective against sars-cov-2 because it forms a stable complex with iron, scavenging ironmediated hydroxyl radical formation and acting as an antiviral (4). what is more, iron chelators can reduce the availability of cellular iron involved in the replication of rna viruses such as west nile virus, hiv, and hepatitis c virus, a property that could be used in the treatment of covid -19 infections (17). the chelator deferasirox has a different effect, binding cytosolic iron discharged from ferritin (5). lactoferrin, a glycoprotein that is part of the body's natural immunity, is one of the potential naturally occurring iron chelators. it is produced by exocrine glands and neutrophils and found in human milk and all secretions. it has a variety of therapeutic effects. apart from iron binding and effect on the immune system, it also diminishes inflammation by affecting the formation of cytokines and ros, thus reducing iron overload. it also inhibits the joining of heparan sulfate proteoglycans, which prevents viruses from cell entry (40). iron chelation therapy in covid-19 as more research indicates that endothelial inflammation is an important pathophysiological mechanism responsible for the multiorgan involvement and organ failure in sars-cov-2 infection, many researchers believe that iron chelators may prove useful in improving the systemic manifestations of covid-19 (41). experimental studies in animals with bleomycininduced pulmonary fibrosis, in which fibrosis and worsening lung function are associated with increased iron aggregation in the lungs, have shown that iron chelator therapy is beneficial (42). due to the lack of adequate therapy, an increasing number of investigators are suggesting that targeted iron therapy may help treat the more severe forms of covid-19, as iron is likely required for viral replication and functions of sars-cov-2 (5). previous research and findings have shown that iron chelation may have an effect on proinflammatory cytokines and free radicals, which are closely related with severe covid-19 disease and may lead to tissue destruction, with acute lung injury and ards being the most severe outcomes. because of all these factors, iron chelators represent a potential covid-19 treatment (5). iron chelators could alleviate ards and contribute to the control of sars-cov-2 through several mechanisms: reduction of iron attainability, inhibition of viral multiplication, increase in the titer of neutralizing antiviral antibodies and b cells, prevention of endothelial inflammation, and inhibition of pulmonary fibrosis and lung decay by reducing pulmonary iron accumulation (41). as mentioned before, iron chelator dfo could be useful as a potential therapy for covid-19 infection because it reduces the replication of some rna viruses, as shown by in vitro studies, and also reduces the availability of iron in serum and body tissues, which could prevent pulmonary fibrosis after covid-19 infection (39). in vitro, it also lowers levels of il-6 seemedj 2022, vol 6, no 2 iron chelation therapy in covid-19 infection 80 southeastern european medical journal, 2022; 6(2) and endothelial inflammation, which could reduce the severity of covid-19 infection and multi-organ damage and failure (39). in a mouse model, preconditioning with dfo was shown to protect the lungs from mechanical ventilation damage by reducing ros formation in mitochondria and macrophages (43). there is only one study of 25 patients that evaluated the effect of tocilizumab and an adjuvant iron chelator in severe covid-19 pneumonia and whether the prescribed therapy would reduce mortality (44). eleven patients received therapy with tocilizumab and the adjuvant iron chelator deferasirox and over 80% had a favorable outcome. the therapy proved to be a good option for patients with significant hyperferritinemia and severe covid-19 disease. two trials are currently underway to check the efficacy and safety of dfo compared to the standard of care or tocilizumab in patients with covid-19 (nct04333550, nct04361032), the results of which are eagerly awaited (5). vlahakos et al have proposed possible therapeutic guidelines for iron chelator therapy (45) several parameters indicative of patient deterioration would be monitored (e.g., oxygen demand ≥60%, ferritin levels ≥ 1000 ng/ml and crp level > 10-fold above baseline, platelets < 100 000 × 109/l and lymphocyte counts < 1000 × 109/l) and their deterioration would indicate progressive severity of covid-19 infection and predict the need for more aggressive critical treatment. it has been suggested that oral iron chelator therapy could be administered 10-14 days after the onset of severe covid-19 infection. iron chelators have been successfully used for half a century to treat diseases with excessive iron accumulation (45). it is possible that intravenous iron chelator therapy may provide sufficient and rapid lowering of plasma iron levels to relieve cytokine storm in patients with severe covid-19 infection in icu. in moderate cases, oral chelators can prevent the development of a severe inflammatory response. scientists all over the world agree that treatment of patients with covid-19 infection should begin as soon as possible and at the appropriate dose. however, it is essential to conduct adequately powered randomized trials before using iron chelators in patients with severe covid-19 infection (45). in addition to iron chelators, hepcidin antagonists could be used as a potential therapy to lower iron levels instead of iron chelators in the supportive care of covid-19 in the future, since all infections trigger inflammation that increases hepcidin levels as the main regulator of iron, causing anemia. it is also known that ferritin formed during inflammation contains less iron than normal ferritin (39,46,47). in addition, cytokines are overexpressed during covid-19, leading to an increase in hepcidin levels (17). dfo decreases the level of il-6, an important inflammatory mediator that triggers a cytokine storm. in addition, there is evidence that the other pharmacological benefit of dfo is the downregulation of hepcidin (39). it has been observed that replication of coronaviruses in iron-deficient cells is suboptimal compared to iron-rich cells (2). some encouraging in vitro studies on the effects of the naturally occurring iron chelator lactoferrin on sars-cov and on sars-cov-2 viruses have shown that lactoferrin inhibits the initial phase of viral infection (40,42,48). iron chelator therapy and its beneficial effects on pneumonia and secondary fibrogenesis suggest that iron chelators should be taken into account to improve the long-term outcome and survival of patients with covid-19, especially those with severe covid-19 infection (5). some of the known iron chelators, such as dfo, deferasirox, and deferiprone, as well as the natural iron chelator lactoferrin, may be efficient in the therapy of covid-19 (4). conclusion according to the literature, iron chelator therapy could have a number of beneficial effects in patients with covid -19 infection, especially in severe forms of the disease, without causing harm in severe covid-19 patients. unfortunately, there are currently not enough adequate randomized prospective trials to confirm the benefits of iron chelator treatment, and the current evidence base is poor. several seemedj 2022, vol 6, no 2 iron chelation therapy in covid-19 infection 81 southeastern european medical journal, 2022; 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(2021). the in vitro antiviral activity of lactoferrin against common human coronaviruses and sars-cov-2 is mediated by targeting the heparan sulfate coreceptor. emerging microbes & infections 2021; 10(1):317-330 doi:10.1080/22221751.2021.1888660 author contribution. acquisition of data: ikk; at, jk administrative, technical or logistic support: ikk; at, jk analysis and interpretation of data: ikk; at, jk conception and design: ikk; at, jk critical revision of the article for important intellectual content: ikk; at, jk drafting of the article: ikk; at, jk final approval of the article: ikk; at, jk guarantor of the study: ikk; at, jk obtaining funding: ikk; at, jk provision of study materials or patients: ikk; at, jk statistical expertise: ikk; at, jk seemedj 2022, vol 6, no 2 trefoil factor 3 protein and sepsis 44 southeastern european medical journal, 2022; 6(2) review article trefoil factor 3 protein and sepsis – a review 1 iva bazina 1, mirela baus lončar 1 1 department of molecular medicine, ruđer bošković institute, zagreb, croatia *corresponding author: iva bazina; ibazina@irb.hr received: jul 26, 2022; revised version accepted: nov 1, 2022; published: nov 28, 2022 keywords: sepsis, trefoil factor 3 (tff3), biomarker, intestinal mucosal barrier disruption, serum tff3 abstract sepsis is one of the most common causes of death in hospitalized patients. disruption of intestinal barrier homeostasis is one of its main hallmarks. trefoil factor family proteins are known for their role in protecting and repairing the intestinal mucosa. it has been repeatedly shown that the tff3 protein is involved in maintaining the intestinal barrier. for that reason, it has been studied in the search for objective measures to predict the onset or outcome of sepsis. several studies have been performed on rodent sepsis models and on sepsis patients, both children and adults. from the limited research available to date, it appears that tff3 is involved in the pathogenesis of sepsis, but the exact mechanism is not yet clear. its potential as a sepsis biomarker has so far been low, but more extensive studies on its role in predicting disease severity and outcome, as well as organ dysfunction, may lead to finding specific patient groups or sepsis stages for which it would be suitable. (bazina i, baus lončar m. trefoil factor 3 protein and sepsis. seemedj 2022; 6(2); 44-53) seemedj 2022, vol 6, no 2 trefoil factor 3 protein and sepsis 45 southeastern european medical journal, 2022; 6(2) sepsis sepsis is one of the leading causes of death in hospitalized patients worldwide [1]. sepsis is a “life-threatening organ dysfunction caused by a dysregulated host response to infection”, as defined by the third international consensus definitions for sepsis and septic shock (sepsis3) in 2016 [2]. since most of the research presented here was conducted before 2016, it is important to note the changes made by sepsis3. earlier definitions stated that sepsis is a consequence of the host’s systemic inflammatory response syndrome (sirs) to infection. when this is complicated by organ dysfunction, it is referred to as severe sepsis, which can progress to septic shock. sepsis-3 considers this continuum misleading, the criteria of sirs nonspecific, the term severe sepsis redundant, and defines septic shock as a subset of sepsis in which the underlying circulatory and cellular/metabolic abnormalities are so profound as to significantly increase mortality [2]. septic conditions can be caused by or lead to alterations in intestinal homeostasis [3]. the gastrointestinal tract is under perpetual pressure from potentially destructive agents, such as chemicals, drugs, bacteria, and their products [4]. the intestinal epithelium, a single layer of cells, forms a selective barrier to the external environment, and is critical in protecting the intestinal mucosa from luminal contents [4]. alterations in homeostasis of the intestinal barrier can lead to increased production of proteolytic enzymes in the intestine, changes in composition of the mucus layer, epithelial apoptosis, increased permeability of epithelial cells, and inflammatory signaling [5–7]. gut barrier dysfunction in combination with disturbed balance of the microbiome and immune response can lead to remote injury and the development of multiple organ dysfunction syndrome (mods), and eventually death [3,8– 10]. trefoil factor family proteins the trefoil factor family owes its name to a characteristic trefoil-like structure consisting of six disulfide-linked cysteine residues in a 38or 39-amino-acid sequence [11–13]. there are three members of the family: tff1, formerly known as ps2 [14], and tff3, formerly itf [15], have a single trefoil domain, while tff2, formerly psp [16], has two. the trefoil domain enables their resistance to digestion by proteases [15–17], which is important because they are secreted by goblet cells into the hostile environment of the intestine. minor disruptions of epithelial integrity are common and require prompt intervention in order to restore homeostasis [18]. trefoil factor family proteins play a role in maintaining mucosal integrity, which is supported by the fact that their accumulation has been shown to be increased after a gastrointestinal tract injury [19– 22]. normal repair of the epithelium requires restitution and regeneration, with restitution being the critical first phase required to reestablish epithelial continuity [23]. trefoil factor family proteins have been shown to be important for intestinal epithelial restitution [18,24], with tff3 even being essential [23]. mice lacking tff3 fail to restitute the intestinal mucosal barrier after injury, which makes even minor injuries lethal [23]. the tff3 protein is involved in maintaining the intestinal barrier by reducing intestinal permeability [25–31]. tff3 preserves the integrity of the intestinal barrier by inhibiting apoptosis induced by inflammatory stress [32]. it is possible that tff3 protects the gastrointestinal mucosa through its interaction with mucins [24,33]. sepsis and its course depend on many factors, including the site and type of infection, genetic predisposition, demographic characteristics, underlying conditions, immune system status, disease progression, treatment, etc., which inherently makes sepsis a very heterogeneous disease [34–37]. in search of objective measures that could predict the onset or outcome of such a complex disease, some research, summarized in this article, has been conducted on proteins belonging to the trefoil factor family [38–47], in particular the tff3 protein [38–42,45–47]. seemedj 2022, vol 6, no 2 trefoil factor 3 protein and sepsis 46 southeastern european medical journal, 2022; 6(2) tff3 in rodent models of sepsis since intestinal barrier disruption has a major impact on the development of sepsis, jiang et al. studied the changes in the intestinal mucosal immune barrier in septic rats induced by cecal ligation and puncture (clp) [38]. clp is the most widely used technique for inducing sepsis in animals [48]. the intestinal mucosa of septic rats exhibited lower tff3 mrna expression compared with control groups. the expression levels decreased with time, namely 3, 6, or 12 hours after induction of sepsis. in parallel with the gradual decrease in tff3 expression, apoptosis increased with time and was higher than in control animals. under these conditions, bacterial dna in the blood was increased, i.e., bacterial translocation increased [38]. later, a group from the same hospital performed the clp experiment on mice [39]. this time, they divided the animals into 6-hour, 24-hour, and 48hour groups, depending on how long after induction of sepsis they were sacrificed, and they measured tff3 protein accumulation. interestingly, the tff3 protein level was slightly increased at the 6-hour time point compared with the control group, while the other two time groups showed a significant decrease in the tff3 protein level. in addition, a significantly lower number of goblet cells was observed in all three groups of treated animals compared with the control group. the researchers did not observe rapid repair of intestinal disruption, which they suggest could be explained by the reduction of tff3, along with a reduction in goblet cell migration to the site of injury and tgf-β1 accumulation, as well as an increase in pro-inflammatory cytokines and pro-apoptotic caspase-3 protein levels [39]. using an lps (bacterial lipopolysaccharide) induced sepsis model in rats, shi et al. [40] demonstrated a delay in intestinal mucosal repair during sepsis. they focused on the molecular mechanism through which protein disulfide isomerase a1 (pdia1) affects the formation of tff3 dimers and how they change during sepsis; they hypothesized that pdia1 and tff3 dimer levels are important contributors to intestinal mucosal destruction and lack of repair, and that pdia1 is the critical catalyst for tff3 dimerization. they confirmed a close correlation between pdia1 and tff3 dimerization using several methods, including protein modeling. they indicated that tff3 monomers are unlikely to dimerize in the absence of pdia1. they noted a decreasing trend in the expression of tff3 (monomer and dimer) and pdia1, as well as impaired pdia1 function in both rat and cell models of sepsis, suggesting that a lack of both may be an important factor in intestinal mucosal damage and impaired repair in sepsis, as impaired pdia1 function may lead to decreased formation of tff3 dimers, ultimately resulting in more intestinal damage and less repair. tff3 in pediatric patients with sepsis in search of a new objective diagnostic marker for gastrointestinal failure, a pediatric intensive care unit in china studied serum tff3 levels of children with mods, where one group had gastrointestinal failure (gif), and the other did not [41]. tff3 levels were associated with both gif and mortality. serum tff3 levels were nearly ten times higher in children with mods and gif than in children with mods and without gif, and they increased over time. in the group without gif, serum tff3 levels remained the same over time and were similar to those of the healthy control group. a total of 71% of the children with mods, gif, and high tff3 died, compared with 32% of the children with normal tff3 and without gif. this suggests that early measurement of serum tff3 levels in hospitals may serve as a prognostic factor, but, as the authors themselves note, the levels should be measured over a longer period in order to see exactly how they change. it should also be noted that this study was conducted in only one hospital in china; it would need to be confirmed in other facilities. a group from brno [42] found that the serum tff3 protein level can distinguish children with sirs, sepsis, severe sepsis, septic shock, and mods (according to the 2005 international pediatric sepsis consensus conference [49]) from control subjects, but the tff3 level was not adequate for distinguishing between the stages seemedj 2022, vol 6, no 2 trefoil factor 3 protein and sepsis 47 southeastern european medical journal, 2022; 6(2) of septic state prior to the development of severe organ dysfunction. in all patients, the tff3 level was significantly higher compared with the control group, and in mods patients, it was significantly higher compared with the rest of the patients. no temporal effect on tff3 protein level was observed. the same group also examined tff1 [43] and tff2 [44] protein levels in the serum of septic children. they found that tff1 levels did not discriminate between different septic states up to the development of significant septic shock and organ dysfunction, and tff2 levels up to organ dysfunction, but the discriminatory power is too low to serve as a diagnostic tool. both could be used to distinguish septic patients from controls, and tff2 could additionally differentiate survivors from non-survivors, as its levels were significantly higher in non-survivors [43]. interestingly, in a study focusing on biomarkers for distinguishing necrotizing enterocolitis (nec) from septic bowel manifestations, no difference was found between preterm infants with sepsis and control groups [46]. nec patients had significantly higher tff3 levels than septic patients, and no association was found between tff3 and nonspecific systemic inflammation indicators, which suggests that there exists only an association with intestinal damage, rather than the systemic inflammatory response [46]. in addition, tff3 has been proposed as a potential new therapy for nec because it was shown that treatment of rat nec models with tff3 alleviated the intestinal tissue injury [45]. tff3 in adult patients with sepsis a study was conducted on adult intensive care unit (icu) patients diagnosed with sepsis [47]. the severity of the condition was determined using the apache ii (acute physiology and chronic health evaluation) score [50], and the extent of organ dysfunction was determined using the sofa (sequential organ failure assessment) score [51]. the elisa assay was used for measurement of septic patients’ tff3 protein levels; they were measured on admission to the icu and after 3 days of treatment in the icu. serum tff3 levels were significantly higher in septic patients than in control subjects, and they increased with time. patients with higher apache ii scores had increased tff3 concentrations, as did patients with higher sofa scores. patients with septic shock, which is defined as severe sepsis complicated with refractory arterial hypotension requiring fluid replacement and vaso¬pressors, had higher tff3 levels than those with uncomplicated sepsis. while serum tff3 levels on admission were only slightly higher in patients with a fatal outcome, tff3 levels were significantly higher three days later in nonsurvivors compared with survivors. high tff3 levels correlated with renal dysfunction, systemic inflammation, long icu stay, and higher mortality. overall, these results suggest that high serum tff3 levels are associated with the severity of organ dysfunction and a worse prognosis for the septic patient. the researchers suggest that tff3 could serve as a prognostic biomarker in the early stages of sepsis, but a larger-scale study is needed first. in a different study [52], serum tff3 protein levels were determined in patients with abdominal sepsis, postoperative gastrointestinal patients, and healthy volunteers. tff3 levels were elevated in patients with abdominal sepsis compared with healthy volunteers, as well as postoperative gastrointestinal patients (no differences were found in the latter two). in contrast to the study presented above [47], tff3 protein levels were maintained for four days in sepsis patients. meanwhile, serum tff3 levels in the postoperative group increased on day 2 and reached sepsis patients’ levels on day 4 post-op, suggesting that plasma tff3 levels increase in response to gi injury of a larger extent or longer duration. tff3 was higher in patients belonging to both the sepsis and the post-op group who were admitted with shock than those without shock. they found markedly increased tff3 levels in patients with three or more organs in failure, and a correlation with renal dysfunction. tff3 levels were higher in abdominal sepsis patients with a fatal outcome; however, tff3 levels at admission were not a good predictor of survival. seemedj 2022, vol 6, no 2 trefoil factor 3 protein and sepsis 48 southeastern european medical journal, 2022; 6(2) studies conducted on human subjects are summarized in table 1. table 1. summary of studies regarding tff3 in the context of sepsis conducted so far on human subjects patient subgroups controls method main findings of the study biomarker potential ref. pediatric patients mods patients with or without gif healthy controls elisa of serum tff3 higher serum tff3 was associated with gif and with mortality. early measurement of serum tff3 levels may prove useful as a prognostic factor. [41] patients with sepsis, severe sepsis, septic shock, and mods patients undergoin g surgery without signs of infection quantitative enzyme immunoass ay of serum tff3 all septic patients had higher serum tff3 levels compared to controls with no signs of infection. mods patients had higher tff3 levels than other patient groups. higher tff3 levels were associated with higher mortality. tff3 levels could not differentiate between various septic conditions in patients until a marked organ dysfunction developed. [42] adult patients patients with sepsis and septic shock healthy blood donors elisa of serum tff3 tff3 concentrations were much higher in sepsis patients than in healthy controls. high serum tff3 levels were associated with the severity of sepsis and mortality. tff3 levels were higher in septic shock than uncomplicated sepsis patients. serum tff3 might serve as a potential prognostic biomarker in the early phase of sepsis. [47] abdominal sepsis, postoperativ e gastrointesti nal patients healthy volunteer s luminex multiplex assay of serum tff3 tff3 levels were elevated in patients with abdominal sepsis compared with healthy volunteers, as well as postoperative gastrointestinal patients. tff3 was higher in patients belonging to both the sepsis and the post-op group who were admitted with shock than those without shock. tff3 levels were higher in abdominal sepsis patients with a fatal outcome. tff3 levels at admission were not a good predictor of patient survival. [52] seemedj 2022, vol 6, no 2 trefoil factor 3 protein and sepsis 49 southeastern european medical journal, 2022; 6(2) the potential of tff3 as a biomarker for sepsis research to date has only scratched the surface of the issue of tff3 in the context of sepsis. although the exact mechanisms and details remain to be elucidated, it is evident from both rodent and human data that tff3 is involved in the pathophysiology of sepsis. in rodent models, lower intestinal tff3 levels were associated with septic conditions and decreased intestinal repair, while in humans, higher serum tff3 levels correlated with disease severity. it is difficult to compare the currently available human and rodent data since they are scarce, describe different things, and so far do not offer any mechanistic explanations. a link between tff3 and sepsis possibly lies in sepsis-induced dysfunction of the intestinal barrier. intestinal permeability and apoptosis [9], as well as both proand anti-inflammatory cytokine production [53] are the hallmarks of sepsis. tff3 is essential for intestinal epithelial restitution [23], and it preserves the integrity of the intestinal barrier by inhibiting apoptosis induced by inflammation [32]. the expression of tff3 can be regulated by inflammatory signals and vice versa [54]. the association between tff3 activation and some known interleukins produced in sepsis, such as il-6 [55,56], il-10 [57], il-8 [58,59], tnf α [60], ifn γ, and il-12 [61], was previously reported. perhaps a balance between cytokine and tff3 activation in sepsis determines the progression and outcome of the disease. the major limitation of sepsis research conducted so far were the small and demographically homogeneous patient groups for such a heterogeneous disease. further and more extensive studies are needed to explore the molecular role of tff3 in the pathogenesis of sepsis, the mechanisms of intestinal damage and repair, the immune response, apoptosis, and the temporal evolution of its effect. it remains to be seen whether sepsis promotes tff3 production or whether tff3 promotes sepsis progression, and at what stage of the disease it makes the greatest contribution. based on the information available to date, tff3 is not a good biomarker for sepsis in general, but further research into its potential for predicting disease severity and outcome, as well as organ dysfunction, may lead to finding specific patient populations or stages of sepsis for which it is appropriate. acknowledgement. this paper was supported by the croatian science foundation grant ip-06-2016-2717 and the european structural fund 2014–2020 as financial support for the phd student i. bazina. disclosure funding. none. competing interests. none to declare. references 1. rudd ke, johnson sc, agesa km, shackelford ka, tsoi d, kievlan dr, colombara d v., ikuta ks, kissoon n, finfer s, fleischmann-struzek c, machado fr, reinhart kk, rowan k, seymour cw, watson rs, eoin west t, marinho f, hay si, lozano r, lopez ad, angus dc, murray cjl, naghavi m. global, regional, and national sepsis incidence and mortality, 1990–2017: analysis for the global burden of disease study. lancet 2020; 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3(1) review article aortic and cerebral aneurysms: link with genetic predisposition, risk factors, and aortopathies 1 luka švitek * 1, nora pušeljić* 1, željka breškić ćurić 2, kristina selthofer-relatić 3, 4 *first authors 1 faculty of medicine osijek, josip juraj strossmayer university of osijek, croatia 2 department of internal medicine, general hospital vinkovci, croatia 3 department of cardiovascular diseases, university hospital centre osijek, croatia 4 department of internal medicine, general medicine and history of medicine, faculty of medicine, josip juraj strossmayer university of osijek, croatia corresponding author: kristina selthofer-relatić, selthofer.relatic@gmail.com received: march 24, 2019; revised version accepted: may 13, 2019; published: may 31, 2019 keywords: atherosclerosis, aortic aneurysm, genetics, inflammation, intracranial aneurysm abstract routine cardiology practice includes diagnostic algorithms for thoracic aortic aneurysm detection at varying degrees of clinical significance. standard procedures for evaluation and follow up involve screening for standard atherosclerotic risk factors, including hypertension, dyslipidemia, diabetes mellitus, obesity, smoking history and family history without genetic testing, as well as cardiac imaging techniques, such as echocardiography, computed tomography or magnetic resonance imaging. according to the latest reports, thoracic aortic aneurysms can present concomitantly with intracranial aneurysms, although the exact etiopathogenic mechanisms are not yet known. there is evidence that connects these two conditions with genetic predisposition, risk factors, and aortopathies. routine practice does not include screening for other aneurysm locations. this review will highlight existing knowledge in this area and the need for further investigations. (švitek l, pušeljić n, breškić ćurić ž, selthofer-relatić k. aortic and cerebral aneurysms: link with genetic predisposition, risk factors, and aortopathies. seemedj 2019; 3(1); 29-41) seemedj 2019, vol 3, no. 1 aortic and cerebral aneurysms 30 southeastern european medical journal, 2019; 3(1) introduction an aneurysm is defined as a focal dilatation of the arterial wall. the cause and pathogenesis of aneurysms remain unknown. the initial arterial dilatation appears to be caused in part by degeneration of a portion of the arterial wall, often medial elastin, followed by smooth muscle. in recent years, there has been greater evidence regarding the concomitant incidence of intracranial aneurysms (ias) and aortic aneurysms (aas). the prevalence of unruptured ias in the general population is estimated to be 3.2%. the prevalence of aas, both thoracic aortic aneurysms (taas) and abdominal aortic aneurysms (aaas), is around 1%–2% in the general population. the prevalence is higher in older populations at approximately 10% (1, 2). the precise mechanisms of initiation, enlargement, or rupture of ias, taas, and aaas remain unknown. biomechanical factors play fundamental roles in aneurysmatic development. an assessment of rupture potential should include geometry (shape and thickness), mechanical properties (anisotropy and strength), and applied loads (hemodynamic and perivascular), without primarily relying on maximum dimension. early loss of elastin and subsequent loss of smooth muscle, with collagen structure change controlled by cellular responses as a result of hemodynamic and intramural stresses, can initiate thinning of the arterial wall and cause enlargement (3). congenital conditions, such as bicuspid aortic valve (bav) and coarctation of the aorta (coa), frequently occur with dilated aortic roots with a predisposition for aortic or cervical dissection. ias are also more prevalent in patients with bav or coa, implying that ias and aortic pathologies share a common developmental defect. the tunica media of both arteries has the same embryological origin, neural crest cells (nccs), and a similar structure of cross-linked elastin and collagen (4, 5). another important and proposed pathway for aneurysm formation involves endothelial dysfunction and/or injury, inflammation, vascular smooth muscle cell (smc) response, extracellular matrix remodeling, vessel wall ageing, and degeneration with end-stage death (6). these processes are common with atherosclerosis etiopathogenesis to a degree, but their relationship with ias and aas is still unknown. the most recent epidemiological, clinical, and biological evidence suggests that atherosclerosis and ias/taas are more distinct than traditionally thought (7). this review will focus on the concomitant incidence of ias and taas, their etiopathogenesis, risk factors, genetic predisposition, and aortopathies. artery – anatomy, histology, physiology and pathophysiology arteries are the main conductive blood vessels that deliver blood from the heart to all parts of the body. the arterial wall consists of three layers: a) intima – innermost layer, consisting of endothelial cells attached to a basement membrane composed of type iv collagen and laminin; b) media – middle layer, built from smcs embedded in extracellular matrix composed of elastin, collagen, and proteoglycans; and c) adventitia – the outermost layer, consisting of fibroblasts and type i collagen with admixed elastin. the largest arteries contain the vasa vasorum, a network of small vessels that supply large blood vessels (3). the cells and matrix of each layer are built to provide a structural and functional support to blood vessel walls (6). the main difference in the structure of systemic arterial vasculature is the composition of the tunica media. according to structure, arteries can be divided into elastic and muscular arteries. the common carotid artery is an elastic artery, while intracranial arteries are muscular arteries. the absence of an external elastic lamina makes intracranial arteries more vulnerable to aneurysmatic formation and rupture compared to other muscular arteries. each arterial site may possess a different degree of durability and vulnerability as a result of seemedj 2019, vol 3, no. 1 aortic and cerebral aneurysms 31 southeastern european medical journal, 2019; 3(1) variance in pathophysiological signals, such as pressure increase (8). although it may occur in any blood vessel, the most common aneurysms according to location are aneurysms of the brain (circle of willis), taas, and aaas (3). also, arterial bifurcations are locus minoris resistentiae for cerebral aneurysm formations because of hemodynamic stress resulting from oscillations in blood flow (8). according to shape, aneurysms can be classified as saccular or fusiform aneurysms. the pathogenesis of aneurysm formation has not yet been clarified, but it is assumed that initial dilatation is first caused by elastin degeneration in the media, followed by smooth muscle degeneration (3). embryology the vascular system starts its development early in the third week of gestation when the first blood islands first appear in the umbilical vesicle. blood islands are composed of mesodermal cells that are induced by fibroblast growth factor (fgf2) to differentiate into hemangioblasts, which are then stimulated by vascular endothelial growth factor (vegf), so they become endothelial cells and bind into the first blood vessels. this is the process of angiogenesis (9). another way in which blood vessels develop is vasculogenesis, which can best be explained as sprouting from pre-existing ones. the earliest formed primitive blood vessel is the dorsal aorta, which soon after becomes surrounded by smcs originating from the splanchnic mesoderm (10). slightly later in development, primary smcs derived from the splanchnic mesoderm are gradually replaced by a secondary population of smcs originating from nccs (11-13). the thoracic aorta almost fully replaces its primary smcs with neural crestderived smcs (11). conversely, the abdominal aorta retains its mesodermal-derived smcs. smcs derived from nccs tend to produce a higher amount of elastin but at the same time express lower contractility function compared to those of mesodermal origin (14). unique responses of nccs to various cytokines and growth factors are firm evidence of the different embryonic origins of the thoracic and abdominal aortas. neural crest-derived smcs from the thoracic aorta show increased dna synthesis and collagen production by transforming growth factor-β1 (tgf) as well as enhanced proliferation, along with synthetic activity, when stimulated by homocysteine. contrary to that, mesodermal smcs show no response to either of these (7, 8). further support for these findings includes studies showing a causative link between the loss of signaling through tgf-β receptors ι and ιι and development of familial taas and dissection (15-17). the proposed underlying cause for this kind of smooth muscle distribution in the aortic wall is that these neural crest-derived smcs can better endure the higher pulse pressure in the thoracic aorta by laying down more elastic lamellae during development and growth (10). the embryological link between the ascending aorta and the intracranial arteries lies precisely in the neural crest origin of smcs. the cerebrovascular structures of the face and forebrain are derived from pericytes and the musculo-connective wall of arteries from nccs (18). malposition or malfunction during the perturbation of these cells (neurocristopathy) could be a plausible cause for the simultaneous occurrence of aas and ias (19). considering the different embryological origins of smcs, shin et al. showed a site-specific relationship between aneurysms of the aorta and intracranial arteries. ascending aortic aneurysms occur more commonly with aneurysms of the anterior and middle cerebral arteries, in contrast to aaas, which occur more often with internal carotid artery aneurysms (20). besides the development of vascular structures, nccs play an important role in the development of head structures, skin melanocytes, a great part of the peripheral nervous system, some endocrine cells, and other structures (21, 22). waldo et al. have contributed to the knowledge about the importance of nccs in the septation of the truncus arteriosus (12). co-occurrence of pathologies, such as a bav (23), coa (24), aortic root dilatation, cystic medial wall degeneration, and cervical arterial dissection (23) all fall into the category of neurocristopathy phenotypes (23, seemedj 2019, vol 3, no. 1 aortic and cerebral aneurysms 32 southeastern european medical journal, 2019; 3(1) 24). studies have also shown higher incidence of ias in neurocristopathy diseases, such as neurofibromatosis type 1 (25), and diseases that are reminiscent of neurocristopathies, such as moyamoya disease, fibromuscular dysplasia, and cervical dissection (26). through these types of head, neck, and heart pathologies, the importance of the participation of nccs in vascular remodeling is emphasized. genetic predisposition given the knowledge of the common embryonic origin, numerous studies have focused their genetic research on diseases related to neurocristopathy phenotype (table 1, figure 1). table 1. gene mutations and clinical presentation on ias2, taas3 and aaas4 mutations clinical repercussion molecular level mutations in the genes encoding the tgfbr1 and tgfbr2 receptors loeys-dietz syndrome tgf-β2 pathway smad3 gene aneurysm osteoarthritis syndrome tgf-β1 pathway (a gene encoding a downstream signaling mediator tgf-β via the tgfbr1 and tgfbr2 receptors) col3a1 gene ehlers-danlos syndrome type iv tgf-β1 pathway (a gene for the collagen type iii pro α-1 chain) pkd1 gene (85%), pkd2 gene (15%) autosomal dominant polycystic kidney disease polycystin-1 and polycystin-2, when healthy, likely work together to help regulate cell growth and division (proliferation), cell movement (migration), and interactions with other cells 3p24–25 and 5q for gene tgfbr2 and cspg2 gene possible loci for ias3 and taas4 tgf-β1 pathway, pathology of the arterial wall 4q32–34 effect on aneurysms of the abdominal aorta and intracranial vessels 11q24 significant role in all 3 sites of aneurysm formation 19q effect on aneurysms of the abdominal aorta and intracranial vessels 9p21, 2q33 found in patients with ias2 and aaas5 18q11 and 15q21 strong connection of ias2 and taas3 with aaas4 1 transforming growth factor β 2 intracranial aneurysms 3 thoracic aortic aneurysms 4 abdominal aortic aneurysms seemedj 2019, vol 3, no. 1 aortic and cerebral aneurysms 33 southeastern european medical journal, 2019; 3(1) figure 1. correlation of ias with neurocristopathy diseases pathology in the septation of heart outflow tracts, such as bav, coa, and dilated aortic root, was shown to co-occur with cerebral aneurysms (24, 27, 28). as evidence for the same pathological origin, a study done by rosenquist et al. showed impaired initiation of elastogenesis in the great vessel walls after the ablation of nccs (29). considering the existing knowledge in embryology about the importance of tgf-β signaling in the development of arterial walls, it is easy to understand that diseases with defects in the tgf-β pathway show a tendency of aneurysm development in multiple vascular beds. loeys-dietz syndrome (mutations in genes encoding the tgfbr1 and tgfbr2 receptors) (30), aneurysm-osteoarthritis syndrome (mutations in smad3, a gene encoding a downstream signaling mediator tgf-β via the tgfbr1 and tgfbr2 receptors) (31, 32), and ehlers-danlos syndrome type iv (mutations in the gene for the collagen type iii pro-α-1 chain [col3a1]) are prone to forming both taas and ias (33). nurmonen et al. showed a higher occurrence of multiple ias in patients with autosomal dominant polycystic kidney disease. likewise, in terms of age of occurrence of aneurysmal subarachnoid hemorrhage, in people with ias it occurs 10 years earlier than in the general population (34). a study of 669 patients with fibromuscular dysplasia showed a significantly higher prevalence of ias (35). linkage studies of the entire genome carried out on predefined loci for three sites of aneurysm formation (intracranial, abdominal, and thoracic aorta) found five overlapping chromosomal regions at 3p24-25, 4q32-34, 5q, 11q24 and 19q. 3p24–25 and 5q for the cspg2 gene are possible loci for ias and taas. 3p24–25 is the locus for the tgfbr2 gene, which was already mentioned in relation to diseases affecting tgf-β signaling, while cspg2 seems to have an effect on the pathology of the arterial wall as well. for the other regions, there is no known candidate gene yet, but 11q24 seems to have a significant role in all three sites of aneurysm formation, while 4q32–34 and 19q have an effect on aneurysms of the abdominal aorta and intracranial vessels (36). in 2016, a mega-analysis of four previously published aneurysm cohort studies was performed (two intracerebral aneurysm cohort studies from the netherlands and finland, an aaa cohort study from the netherlands, and a taa cohort study from the united states). it detected a single nucleotide polymorphism at four loci (9p21, 2q33, and two chromosomal regions, 18q11 and 15q21) which showed a strong connection between ias and taas with aaas (37). in a study by shin et al., while searching for evidence of innate aortopathy in patients with ias, the scientists noticed that short stature was common among the respondents (24). the aforementioned nccs have a number of roles outside the development of the vascular system, including a role in the development of the pituitary, which can have an effect on the growth of bone and cartilage (21, 38). further research on other potential, unknown causes could highlight this problem and provide new insights in this area. atherosclerosis atherosclerosis correlates with arterial aneurysmatic occurrence. it is still unknown if it is a causal relationship, or if they share common risk factors (39). most studies agree that seemedj 2019, vol 3, no. 1 aortic and cerebral aneurysms 34 southeastern european medical journal, 2019; 3(1) atherosclerosis is mainly correlated with aaas, but not with taas and ias (3, 39-41). there is a shared genetic background for both aaas and atherosclerosis (39, 42, 43), as well as common risk factors, including hypertension, obesity, smoking, hdl, family history, and thrombosis (2, 20, 42, 44-51). on the other hand, some markers differ, such as ldl (no correlation with aaas) and diabetes (negative correlation with aaas) (42, 50, 51). there is no conclusive evidence about the correlation between the development of ias and atherosclerosis. rouchaud et al. showed that there is no correlation between atherosclerosis and ias (46). wang et al. even asserted that atherosclerosis is a protective factor against ia rupture (52), while hokari et al. suggested that middle cerebral artery aneurysms positively correlate with hypertension and cerebrovascular disease, which are common comorbidities in patients with atherosclerosis and have a higher incidence in stroke patients (41). another type of ias, paraclinoid aneurysms, were also the subject of the same study. paraclinoid aneurysms are defects of an internal carotid artery wall that are located between the superior part of cavernous sinus and the entrance for the posterior communicating artery (53), detected with three dimensional time-of-flight magnetic resonance angiography (3d tof mra) in stroke patients, as well as in healthy adults volunteering for asymptomatic brain disease evaluation (41). the results showed that paraclinoid aneurysms have a lower incidence in stroke patients than in healthy research participants (41). likewise, paraclinoid aneurysms are more affected by hemodynamic stress and unchangeable risk factors than middle cerebral artery aneurysms (54). these results might not mean that ias are “immune to atherosclerotic risk factors”, but just that they are not equally affected regarding the localization. inflammation genetics, hemodynamic changes, and environmental factors are the main factors in aneurysm development. in recent years, there has been considerable evidence for inflammation as a leading factor in the pathogenesis of ias (6, 39, 55). initially, in response to hemodynamic stress, development of endothelial dysfunction is followed by an inflammatory reaction. macrophages release proinflammatory cytokines that lead to recruitment of additional inflammatory cells and release matrix metalloproteinases (mmps), which cause degradation of the extracellular matrix and activate other proteinases (55). according to a study by aoki et al., mice with lower expression of monocyte chemoattractant protein-1 (mcp-1) and decreased accumulation of macrophages had a lower risk of developing cerebral aneurysms (56). likewise, according to another study by aoki et al., the application of mmp inhibitor in rats reduced the progression of ias (57). mmps cause apoptosis of smcs, which are mostly concentrated in the media, leading to the thinning of this layer. tumor necrosis factorα (tnf-α) modulates smcs by inhibiting collagen synthesis, which is a predisposition for aneurysm formation, progression, and rupture (55). t cells, mast cells, and cytokines also participate in inflammation and aneurysm formation (8). tnf-α and interleukin-6 (il-6) are the best explored cytokines involved in the development of inflammation and aneurysm formation. complement activation has been found in patients with ias, but it is still necessary to explain how it contributes to aneurysm formation (58). according to a study by shikata et al., intestinal microbiota in mice can affect the formation of ias by modulating inflammation through depletion of macrophage infiltration and lowering of inflammatory cytokine levels in blood vessel walls (59). understanding all aspects of the role that inflammation plays in the formation of aneurysms is essential because there are many sites that can potentially present target sites for pharmacological treatment of aneurysms. some agents were studied for inhibiting aneurysm seemedj 2019, vol 3, no. 1 aortic and cerebral aneurysms 35 southeastern european medical journal, 2019; 3(1) development, such as statins, aspirin, and inhibitor of phosphodiesterase-4 (ibudilast), but their role in the treatment has not yet been conclusively proved (6). aorthopathy according to recent evidence, there are correlations between ias and aas, especially taas, which are based on an aortopathy of a different cause (figure 2) (4, 5, 20, 46, 47, 60, 61). figure 2. algorithm for relation of ias with risk factors and aortopathies regarding the prevalence of ias, studies have shown similarities in groups of patients already known to suffer from aas (4, 5, 20, 46). rouchaud et al. conducted a study on 1081 patients with aas and found the prevalence of ias to be 11.8% (46). likewise, shin et al. carried out a study on 611 patients with aas and showed the prevalence of ias to be 11.6% (20). in addition, jung et al. searched for ias in patients with dissecting aas, and the prevalence was 13% (4). furthermore, research by kuzmik et al. conducted on 212 patients with taas showed a prevalence of 9.0% for ias (60). these results show a much greater prevalence of ias in patients with aas, with the rate of ias four times higher in patients who also have aas (4, 20, 46) than in the general population. taas and ias do not correlate with atherosclerotic risk factors (2, 20, 45-49). research by rouchard et al. showed that coronary artery disease and hyperlipidemia actually lowered the odds of ias (46). multiple aneurysms in patients with ias occur in 15–45% (5, 62, 63) of cases, which may suggest that another underlying cause increases susceptibility to aneurysms. another study by shin et al. focused on congenital aortopathy. they identified the presence of bav and coa (5), already known conditions that are connected with greater seemedj 2019, vol 3, no. 1 aortic and cerebral aneurysms 36 southeastern european medical journal, 2019; 3(1) prevalence of ias (64-66). these are conditions that are usually associated with a dilated aortic root (5). the study showed that a group of people older than 55 who had large ias (more than 7 mm in diameter), multiple aneurysms, or ruptured aneurysms had a significantly higher prevalence of dilated aortic roots (5). another group in this study consisted of patients without the high-risk factors mentioned for the first group (5). the second group had a significantly higher percentage of cerebrovascular risk factors, such as hypertension, diabetes mellitus, and hyperlipidemia, as well as strokes (5). similarly, a study by egbe et al. conducted on 678 patients with bav showed an ia prevalence of 7.7% (66). in this study, the prevalence of ias in patients with both bav and coa was 12.9% (66). in addition, the presence of right-left cusp fusion in patients with bav was found to be a significant risk factor for developing ias (66). a study by schievnik et al. conducted on patients with bav found that 9.8% of patients had ias (65). in a study of 117 patients with coa, curtis et al. found the prevalence of ias to be 10.3% (48). a similar study by connolly et al. conducted on 100 patients with coa determined that 10.0% of patients had ias (64). aortic dissection is another condition that has been studied to evaluate its co-occurrence with ias. as mentioned above, a study by rouchaud et al. conducted on 71 patients found the prevalence of ias to be 13% in patients with dissections (4). that is about four times higher than in the general population (4). these patients had hypertension and a history of cerebrovascular disease in numbers significantly higher than in the control group (4). this study found a significant correlation between the presence of ias and the arch involvement seen in aortic dissection (4). an additional important variable with influence on ia formation in patients with aas is gender (4, 5, 20, 46). a few studies have shown that women are more prone to aneurysms compared to men (20, 46, 66), but this finding requires further research. conclusion according to available data in the areas of genetics, pathophysiologic findings, risk factors, and aortopathies, there are strong relationships and predispositions for concomitant aneurysmatic formation in different loci. the embryological link between the ascending aorta and the intracranial arteries lies in the origin of smcs or nccs. at the developmental level, the septation of heart outflow tracts, such as a bav, coa, and dilated aortic root, is in direct relation with ias as a result of impaired initiation of elastogenesis. atherosclerosis is not a unique risk factor for ias and taas, but is directly related to aaas as well. there are still many unanswered questions in this area in regard to the following: a) genetic predisposition; b) structural and mechanical properties: the extensive differences in histology and pathophysiology, the cause of different shapes of ias (saccular) and taas (fusiform), and difference between vessel wall structures; c) epidemiological risk factors between these three conditions (ias, taas, and aaas); d) gender differences; e) the impact of ageing; f) the need for further research and gene detection. co-occurrence of aortic and cerebral aneurysms exists in multiple conditions. it is not likely that a single genetic disorder will be found to explain the co-occurrence of both aneurysm sites. when it comes to explaining this complex dual pathology, the most probable pathophysiological explanation is that several levels of genetic defects, plus a multifactorial environmental effect, result in arterial wall weakening and the formation of aneurysms in multiple locations. acknowledgement. none disclosure funding. no specific funding was received for this study. competing interests. none to declare seemedj 2019, vol 3, no. 1 aortic and cerebral aneurysms 37 southeastern european medical journal, 2019; 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revised version accepted: april 12, 2017; published: april 24. 2017 keywords: hepatitis c infection, treatment, direct-acting antivirals in 1989, there have been numerous advances in medical therapies available for the treatment of hcv infection. the main aim of antiviral treatment is to eradicate the virus, defined as a sustained virologic response (svr), which means undetectable levels of plasma hcv rna for 12 to 24 weeks after completion of therapy (4). svr is associated with decreases in all-cause mortality, liver-related death rates and complications, and hepatocellular cancer rates (5, 6). the first agents available for treatment of hcv were the alfa interferons which resulted in svr rates of approximately 15% (7). the combination of interferon (ifn) and ribavirin (rbv) improved svr rates to 41% for 48-week abstract hepatitis c virus infection (hcv) infects approximately 185 million individuals worldwide. it is a leading cause of chronic liver disease and the primary reason for liver transplantation. the main aim of antiviral treatment is to achieve a sustained virologic response, which means eradication of the virus. the combination of pegylated-interferon and ribavirin was the standard of care for over a decade, despite the long treatment duration and severe adverse effects. the introduction of direct-acting antivirals with pan-genomic properties and excellent tolerance increased rates of svr and shortened the duration of the therapy. furthermore, it allowed clinicians to customize hcv therapy according to important clinical parameters such as hcv genotype and liver fibrosis stage. (jakab j, žulj m, volarić n, tepeš b, včev a. hepatitis c treatment: a review and update. seemedj 2017;1(1);11-18) seemedj 2017, vol 1, no. 1 hepatitis c treatment: a review and update 12 southeastern european medical journal, vol 1, 2017. treatment regimens (8). with the introduction of pegylated-interferon (peg-ifn), svr rates increased to 50% (9). besides its limited effectiveness, the combination of peg-ifn and ribavirin was associated with long treatment duration and frequent and severe adverse effects, but remained the standard of care for over a decade. the introduction of direct-acting antivirals (daas) has revolutionized therapeutic options for hcv infection (10). the first two approved drugs were boceprevir and telaprevir, ns3/4a protease inhibitors, used in combination with traditional dual therapy (11, 12). this strategy managed to increase the rates of svr to 70% (1012), but with greater toxicity, an increased pill burden, drug-drug interactions, low barriers to resistance, and notable side effects (13-15). since 2013, and the introduction of new, more effective daas with pan-genomic properties and excellent tolerance, the treatment of hcv has had a new scenario: increased rates of svr (even up to 100%), shorter therapy duration and less toxicity (16). direct-acting antivirals currently available daas are classified into four categories based on their mechanism of action: non-structural 3/4a serine protease inhibitors, non-structural 5a inhibitors, nucleotide analogue inhibitors of the non-structural 5b polymerase and non-nucleoside inhibitors of the non-structural 5b polymerase (17, 18). these drugs target the replication, polyprotein processing, packaging in nucleocapsid, assembly, and release of hcv infectious particles (3). mutations at different positions in the ns3 protease, ns5b5 polymerase and ns5a protein affect viral susceptibility and the outcome of daa-based therapies. however, the high specificity of daas makes them sensitive to small changes in the viral sequence responsible for antiviral resistance (10). different currently approved interferon-free combination therapies with daas provide synergistic antiviral potency and prevent the development of daas resistance (19). first-generation ns3/4a protease inhibitors the ns3/4a protease is involved in posttranslational processing and releasing of the ns3, ns4a, ns4b, ns5a and ns5b from the hcv polyprotein (20), and it is critical for replication of the virus (21). in 2011, the first generation of ns3/4a inhibitors (boceprevir and telaprevir) was specific for the treatment of chronic hepatitis c, genotype 1, and was used in combination with peginf and rbv. the mechanism of action is the forming of a reversible covalent bond with the ns3/4a active site. limitations to the triple therapy for chc included adverse effects such as gastrointestinal issues, skin reactions and bone marrow toxicity, and low barriers to resistance (22). also, boceprevir and telaprevir are potent cytochrome p-150 inhibitors, resulting in significant drug-drug interaction (12). second-generation protease inhibitors second-wave protease inhibitors offer several advantages over the first generation: improved pharmacokinetics, which allows a once-a-day dosing schedule, more tolerable side-effects, and fewer drug-drug interactions (23). ns5a inhibitors because of its critical involvement in viral replication and assembly (27), ns5a has been identified as a target for viral inhibition. inhibition of ns5a at picomolar concentrations has been associated with significant reductions in hcv rna levels in cell culture-based models (28, 29). ns5a inhibitors have pan-genotypic activity. synergistic inhibition of viral production and an increased barrier to resistance (30) have been accomplished with the use of multiple daas, including an ns5a inhibitor, in cell culture. the exact mechanism of antiviral action of ns5a inhibitors is unknown, and its detailed function remains unclear. available evidence suggests that they have multiple effects, interfering with several functions of ns5a in the hcv life cycle, and disrupt the establishment of replication sites (31). ns5a inhibitors are daclatasvir, ombitasvir, ledipasvir, elbasvir, and velpatasvir. seemedj 2017, vol 1, no. 1 hepatitis c treatment: a review and update 13 southeastern european medical journal, vol 1, 2017. polymerase ns5b inhibitors ns5b is the rna-dependent rna polymerase, and it is next reasonable target for the treatment of hcv. ns5b inhibitors can be classified into non-nucleotide inhibitors (nnis) and nucleotide inhibitors (nis) (32), both of them binding to the ns5b polymerase to terminate replication of the virus. ns5b utilizes the hcv rna genome as a template for rna synthesis. nucleoside analogue inhibitors incorporate into the hcv rna chain leading to chain termination. because of that, ns5b inhibitors commonly show pangenotypic activity with a high barrier to resistance. they are commonly used in combination with other classes with higher barriers to resistance. the most used ns5b inhibitors are sofosbuvir, as a nucleoside analogue inhibitor (33), and dasabuvir, as an example of a non-nucleoside inhibitor. currently available daas and treatment strategies a number of highly effective daas allow clinicians to customize hcv therapy according to hcv-genotype, liver fibrosis stage and previous treatment experience (34, 35). commercialized daas approved by the united states food and drug administration (fda) (4) are listed in table 1. several factors must be taken into account when selecting the suitable therapeutic regimen. clinically, it is necessary to evaluate table 1. the list of currently available daas medicine brand name daa class dosing genotypes sofosbuvir sovaldi nucleotide ns5b polymerase inhibitor 400 mg (one pill) per day genotypes 1, 2, 3, 4 simeprevir olysio ns3/4a protease inhibitor 150 mg (one capsule) per day genotype 1 daclatasvir daklinza ns5a protein inhibitor 60 mg (two pills) once a day genotypes 1, 3 and 4 [9] sofosbuvir + ledipasvir harvoni ns5a inhibitor sofosbuvir (400 mg) plus ledipasvir (90 mg), in a single pill taken once per day genotype 1 [10] ombitasvirparitaprevir/ ritonavir and dasabuvir viekira ns5a inhibitor + ns3/4a protease inhibitor + cyp3a4 enzyme inhibitor + non-nucleoside ns5b polymeraseinhibitor 12.5-75-250-50 mg genotype 1 daclatasvir in combination with sofosbuvir +/ rbv ns5a inhibitor + nucleotide polymerase ns5b inhibitor 60 mg once daily + sofosbuvir 400 mg once daily ± weightbased ribavirin genotypes 1-4 velpatasvir in combinationwith sofosbuvir epclusa ns5a inhibitor + nucleotide ns5b polymerase inhibitor 400 mg of sofosbuvir and 100 mg of velpatasvir once daily genotypes 1-6 seemedj 2017, vol 1, no. 1 hepatitis c treatment: a review and update 14 southeastern european medical journal, vol 1, 2017. prior treatment history, the severity of the liver table 2. treatment strategies in croatia clinical parameters treatment option genotype 1 naive patients mild fibrosis peg ifn +rbv for 24-48 weeks. advanced fibrosis simeprevir/sofosbuvir and peglfn + ribavirin. significant fibrosis, contraindications to ifn therapy, presence of extrahepatic manifestations, hivcoinfection, transplanted patients ifn-free regimens for 12 weeks: ombitasvir, ritornavir-boosted paritaprevir, dasabuvir + ribavirin; sofosbuvir and ledipasvir ± ribavirin; sofosbuvir and simeprevir ± ribavirin. decompensated cirrhosis sofosbuvir and ledipasvir ± ribavirin for 24 weeks. genotype 1 experienced patients relapse or partial responders with f1-f3 pegifn-α + rbv and simeprevir or sofosbuvir for 12 weeks. non-response (regardless of fibrosis); f4 fibrosis (regardless of type of response); tt il-28b genotype; contraindications to ifn therapy; extrahepatic manifestations, hiv-coinfected and transplanted patients ifn-free regimens for 12 weeks: ombitasvir, paritaprevir and ritonavir, dasabuvir ± ribavirin; sofosbuvir and ledipasvir ± ribavirin; sofosbuvir and simeprevir ± ribavirin. decompensated cirrhosis sofosbuvir and ledipasvir with ribavirin for 12 weeks, or 24 weeks without ribavirin. previously treated with pegifn-α + rbv + pi sofosbuvir + ledipasvir ± ribavirin for 12 weeks. genotype 4 the same recommendations as for genotype 1, with the exception of fixed combination of ombitasvir, paritaprevir and ritonavir, which is used without dasabuvir. genotype 2 treatment naive with f1-f3 fibrosis pegifn-α and rbv for 24 weeks. treatment naive with f4 fibrosis, treatment experienced regardless of fibrosis stage, contraindications to ifn, extrahepatic manifestations or hiv coinfected patients, transplanted patients sofosbuvir and ribavirin for 12-20 weeks (depending on cirrhosis). genotype 3 treatment naive with f1-f3 fibrosis peg ifn and ribavirin for 24 weeks. treatment naive with f4 fibrosis, treatment experienced pegifn-α, rbv and sofosbuvir for 12 weeks. f1-f3 fibrosis, contraindication to ifn sofosbuvir and ribavirin. f4 fibrosis, contraindication to ifn therapy sofosbuvir, ledipasvir and ribavirin for 24 weeks or sofosbuvir, daclatasvir and ribavirin for 24 weeks. seemedj 2017, vol 1, no. 1 hepatitis c treatment: a review and update 15 southeastern european medical journal, vol 1, 2017. disease, the degree of hepatic fibrosis, the presence of decompensation in patients with cirrhosis, renal function, the presence of extrahepatic manifestations of hcv, and the other medications the patient is taking. virologically, it is necessary to know the genotype and its subtype, as well as to monitor the viral load at 12 to 24 weeks after therapy is completed and, in some cases, to determine the il28b polymorphism or q80k mutation (36, 37). the highest priority for treatment should be given to the patients with advanced fibrosis, compensated cirrhosis, severe extrahepatic hepatitis, as well as those on the active liver transplant waiting list, and liver transplant patients with recurrence of the infection. patients with high priority for treatment are the non-responders to triple therapy or patients with extrahepatic manifestations of hcv. independently of the disease status, treatment should also be considered for certain subgroups of hcv patients, such as patients at high risk of transmission or women of child-bearing age (34, 35). hcv treatment in croatia the prevalence of hcv in croatia in the general population is low, with the estimated number of hcv patients around 39,000 (38, 39) and higher prevalence rates in prison populations, hiv patients, individuals with high-risk sexual behavior and alcohol abusers. management of hcv infection in croatia is following the european association for the study of the liver (easl) guidelines, croatian guidelines, and the recommendations of the croatian health insurance fund (hzzo). according to the recommendations, the highest priority is given to the patients with significant fibrosis or cirrhosis, extrahepatic manifestations, in patients with hcv recurrence after liver transplantation and in hbv/hcv and hiv/hcv-coinfected patients. priority is also given to individuals at risk of transmitting hcv (injection drug users, people with high-risk sexual practices, women of childbearing age, haemodialysis patients). treatment can be postponed in patients with no or mild liver fibrosis with no clinically significant extrahepatic manifestations. on the other hand, treatment is not recommended in patients with limited life expectancy (35-37, 40, 41). available drugs covered directly by the croatian health insurance fund in 2016 are peg ifn; ribavirin; simeprevir; sofosbuvir, ombitasvir + ritonavirboosted paritaprevir +/dasabuvir; and sofosbuvir + ledipasvir. treatment strategies for patients with different clinical parameters are listed in table 2. conclusion the appearance of direct-acting antiviral drugs has brought significant advances in the treatment of hepatitis c – high tolerability and 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(last accessed march 14th 2017) https://www.ncbi.nlm.nih.gov/pubmed/24984325 https://www.ncbi.nlm.nih.gov/pubmed/24984325 corrigendum: correction of the author's affiliation author radoja ivan added affiliation: ivan radoja1,3, oliver pavlović1,nikica perić1, dunja degmečić2. coital urinary incontinence and female sexual function. seemedj 2017;1(2);11-18. 1 department of urology, university hospital center osijek, osijek, croatia 2 faculty of medicine, josip juraj strossmayer university of osijek, university department of psychiatry, 3 faculty of medicine, postgraduate doctoral study program, josip juraj strossmayer university of osijek published in: seemedj 2019; 3(1):78. seehsj south eastern europe health sciences journal southeastern european medical journal seemedj faculty of medicine osijek josip juraj strossmayer university of osijek j. huttlera 4 croatia – 31000 osijek seemedj@mefos.unios.hr seemedj.mefos.unios.hr editorial dear colleagues, i am happy to present you with the new issue of southeastern european medical journal (seemedj 2018;(2):1:1-58). this is the fourth number in the second year of journal publication and it is devoted to nursing. this issue brings papers from croatian authorsnurses from various fields. the review by mikšić et al. is on the theory of unpleasant symptoms and the concept of nursing support, this theory helps in planning nursing interventions and facilitates healthcare delivery in general. authors batrnek et al. provide original research on comorbidities of posttraumatic stress disorder and alcohol dependence in more than 150 patients, with findings on increased divorce rate and alcohol consumption with the prescribed therapy in this population. two other papers are concerned with the problems of stress in nurses in the surgery intensive care units (pačarić et al, work-related stress and most common stressors for surgical nurses and pačarić et al, emotional control in surgical and intensive care nursing: sociodemographic differences). both papers are original research papers with important results on burdens and management of stress in nurses, while paper by maceković m and prlić n. provides information on the attitudes of health care professionals on the importance of public health nurses’ education. finally, the paper by raza and miedany (uk) brings the comprehensive review on giant cell arteritis and an attempt to identify the standard of care in individuals with suspected giant cell arteritis in a typical district general hospital and to offer a proposed pathway for treatment of the disease. southeastern european medical journal seemedj faculty of medicine osijek josip juraj strossmayer university of osijek j. huttlera 4 croatia – 31000 osijek seemedj@mefos.unios.hr seemedj.mefos.unios.hr it is important to mention the art work at the cover page of this issue. it is a painting by julije knifer “ap xp” from the museum of fine arts in osijek (selected by ms valentina radoš, senior curator). julije knifer (born in osijek) was our fellow citizen, renowned croatian writer and painter. on behalf of editorial board and my own, i warmly greet our readers and invite them to join us in the endeavour of publishing own scientific work in seemedj. sincerely, ines drenjančević, md, phd editor-in-chief southeastern european medical journal (seemedj) seemedj 2023, vol 7, no 1 thermoregulation and exercise 13 southeastern european medical journal, 2023; 7(1) review article the physiology of thermoregulation in exercise: a brief review 1 helena lenasi* 1, janez šijanec 2 1 institute of physiology, faculty of medicine, university of ljubljana, slovenia 2 bežigrad high school, ljubljana, slovenia *corresponding author: helena lenasi, helena.lenasi.ml@mf.uni-lj.si received: mar 20, 2023; revised version accepted: apr 6, 2023; published: apr 30, 2023 keywords: exercise, thermoregulation, skin microcirculation, vasodilation, sweating abstract during physical exercise, the production of heat in the working skeletal muscles increases, imposing heat stress on the body. thermoregulatory mechanisms induce adjustments of cutaneous vascular conductance and thus skin blood flow (skbf), sweating rate, and increased cardiac output to achieve thermal homeostasis. the response depends on the intensity, type, duration of exercise, and environmental temperature: during extreme exercise in a hot environment skbf can attain up to 7 l/min compared to 300 ml/min at rest whereas the sweating rate can reach as high as 4 l/h. due to opposing non-thermal reflexes, the thermoregulatory response of skbf during exercise differs from that at rest: the threshold to induce vasodilation in the skin is shifted to higher body core temperature and the sensitivity of the “skbf to-core temperature” slope is altered. regular training induces better adaptations to physical stress which enable sportsmen to eliminate additional heat more optimally. the review emphasizes physiological mechanisms involved in thermoregulation during exercise and exposes some thoughts regarding the estimation t of the core temperature in humans, as well as some new approaches for an up-to-date assessment of parameters important for appropriate heat dissipation thereby maintaining core temperature. (lenasi h*, šijanec j. the physiology of thermoregulation in exercise: a brief review. seemedj 2023; 7(1); 13-27) seemedj 2023, vol 7, no 1 thermoregulation and exercise 14 southeastern european medical journal, 2023; 7(1) introduction humans as homeothermic organisms need to maintain their body temperature irrespective of relatively high fluctuations in their external or internal environment. during resting conditions, it is mainly external temperature that is fluctuating, whereas, during exercise, the most profound temperature perturbation occurs in the internal environment (1, 2). though, when exercising in extreme environmental temperature conditions, this additionally impacts the human thermoregulatory system (3, 4). thermoregulation encompasses reflex mechanisms enabling that heat elimination from the body equals heat production thus maintaining a constant body core temperature. core temperature is regulated by a classical negative feedback loop including sensors (central and peripheral thermoreceptors), thermoregulatory center and effectors (2, 5). the information on temperature perturbation is relayed by thermoreceptors to the thermoregulatory center located in the central nervous system which compares the sensed value with the more or less flexible reference temperature value (“set-point”) and accordingly switches on and off effector mechanisms to eliminate the perturbation and return the core temperature to the “set-point” value (2, 5). the main effectors include skin arterioles and sweat glands, brown adipose tissue and skeletal muscles. in addition, behavioral changes contribute to thermoregulation (2, 5). heat is eliminated from the body by four distinct physical principles, namely radiation, conduction, convection and evaporation (passive and active). the physiological thermoregulatory mechanisms in response to core temperature perturbation adjust the fractions of the four physical principles according to the body’s needs: during rest, the majority of heat is eliminated by radiation (60%) and only 20% by passive perspiration, while during exercise the majority of heat elimination is achieved by active evaporation and convection (2, 6). physiological principles of thermoregulation during exercise while skeletal muscles consume approximately 1.5 ml oxygen/min/kg body weight in resting conditions, their oxygen consumption might reach up to 150 ml/min/kg during intensive exercise (4, 5, 7), implying additional heat burden that during exercise may exceed 1,000 watt (w) as compared to only 70 w at rest. a thermal load of this magnitude would raise the core temperature by 1oc every 10 minutes if there were no thermoregulatory mechanisms (4). accordingly, thermoregulatory reflexes are activated to eliminate this additional heat from the body to the environment, respectively preserving thermal homeostasis. thermoregulatory center situated in the preoptic area of the anterior hypothalamus, the thermoregulatory center is believed to comprise a “cold-sensitive” and “hotsensitive” area activated by a corresponding temperature perturbation (8–11). yet, the exact anatomic location, the neurotransmitters involved, and the effector pathways are not precisely known in humans as most of the information has been obtained from animal model experiments (8). the center is regarded as an integrator, integrating thermal information from the periphery and core (2): in response to the mismatch between the perturbation and the reference signal, an autonomic involuntary thermoregulation response is mediated by descending projections from the preoptic area, leading to activation of different thermoeffectors, mainly through the sympathetic pathways (9, 12, 13). an important contribution of oxytocin, modulating autonomic and behavioral mechanisms underlying thermoregulation at both central and peripheral levels has been implicated (14). thermoreceptors the thermoregulatory center receives and integrates information on temperature perturbation from the central and peripheral thermoreceptors, respectively (2). central thermoreceptors are located mainly in the seemedj 2023, vol 7, no 1 thermoregulation and exercise 15 southeastern european medical journal, 2023; 7(1) central nervous system (brain, the spinal cord), although other parts, such as blood vessels and skeletal muscles have been implicated (2). however, the most important ones are part of the thermoregulatory center in the preoptic area and anterior hypothalamus (2, 8, 9). the peripheral thermoreceptors are scattered in the skin throughout the body. similarly, as for the central ones, there are two types of anatomically distinct peripheral thermoreceptors: warmth receptors and cold receptors, whose distribution and density differ regarding skin site (2, 5, 9). central thermoreceptors during exercise, the central thermoreceptors receive the information of increased core temperature (in the range of 2ºc to 4ºc) (2, 5) when a certain temperature threshold, usually denoted as a “set-point” value, has been reached, effector mechanisms are activated for heat elimination which subsequently establishes thermal homeostasis (2, 5, 9). the exact physiological background of the set-point has not been known; the role of various “transientreceptor potential” (trp) membrane channels, similarly as for the cold sensing, have been implicated in the activation of these “hotsensitive” neurons (9, 13, 15). it is worth mentioning that the “set-point” has a rather variable value, as for example during fever it is shifted to higher values (2). similarly, the value of the “set-point” changes during exercise, i. e. the system is operating at a higher temperature steady state during exercise as compared to resting conditions (2, 5, 7). moreover, additional factors importantly affect the value of the “setpoint”, such as the circadian rhythm (6, 16), and sex as well as the phase of the menstrual cycle in premenopausal women (17–19), consequently also impacting exercise performance. apart from these reflexes of the negative feedback mechanisms, some of the effector mechanisms are activated before an increase in core temperature, at the very beginning of exercise, denoting a “central command” and assumingly reflecting the anticipatory, feedforward impulses from the prefrontal, supplementary and premotor cortex (5, 11, 20). peripheral thermoreceptors in addition, the hypothalamus also receives information on the temperature changes over the skin surface relayed by peripheral thermoreceptors, specialized free nerve endings embedded in the skin dermis all over the skin surface (1, 2, 5). changes in surface skin temperature are sensed before the core temperature is altered and, in this respect, peripheral thermoreceptors represent a protection mechanism that diminishes too extensive fluctuations of core temperature during resting conditions, (5), and might be regarded as an additional feed-forward thermoregulatory mechanism (2, 5, 9). during exercise, the response differs: peripheral thermoreceptors are thought to modulate the response of the thermoregulatory center during exercise (2, 6, 15, 21). as explained later, at the beginning of exercise, vasoconstriction in the skin is sufficient to cause temporal storage of excessive heat produced by the working muscles inducing an increase in core temperature which is then sensed by the central thermoreceptors, and reflexes for heat dissipation are activated (2, 4, 6). in fact, during exercise when a new steady-state core temperature is established, mean skin temperature decreases during exercise because of the increased evaporative cooling of the skin caused by sweating (2), increasing the temperature gradient between the body surface and the environment, thus making conductiveconvective heat elimination more efficient (2, 5). in a warm environment and when skin temperature is high, the mechanisms of heat elimination are activated at lower core temperature, i.e. the threshold for heat elimination is lowered (3, 4, 11, 22–24), while the threshold is increased in a cool environment when skin temperature is low (25, 26). peripheral thermoreceptors have been shown to have a great potential for adaptability, especially when exercising in a hot environment; nevertheless, the mechanisms of these adaptations have not been fully understood (15, 24, 27). seemedj 2023, vol 7, no 1 thermoregulation and exercise 16 southeastern european medical journal, 2023; 7(1) increased core temperature induces fatigue and might lead to hyperthermia the increased core temperature has been linked to “central fatigue”, which is speculated to represent a protection mechanism from potential brain injury (10, 28, 29). fatigue and exhaustion are warning signs that exercise should be stopped or adjusted to reduce brain temperature increase. brain temperature is determined by its metabolic activity and its perfusion, reflecting the temperature of blood (2, 15, 30). many studies are being conducted on how to reduce brain heating during exercise, accordingly, improving thermoregulatory mechanisms, prolonging exercise duration and improving sports performance. the application of central pre-cooling or whole-body cooling has been advocated, being achieved by drinking cold drinks, applying cooled gel sacks, or wholebody cooling using special cool water-recycling suits (31–33); yet the best regime needs to be defined. as evaporative-heat loss is imminently connected with water loss, the role of an appropriate replacement of fluid and electrolyte could not be overemphasized (1, 15, 34). hyperthermia exaggerates fluid loss, inducing a self-perpetuating, vicious cycle of both hyperthermia and hypovolemia, potentially leading to heat stroke and hypovolemic shock (1, 2, 5, 35). cutaneous circulation is the main effector of thermoregulation during physical exercise in response to increased heat stress during exercise, the thermoregulatory center activates reflexes to transfer additional heat to the body surface from where heat is dissipated into the surroundings, accordingly, establishing a new steady state. besides an increase in cardiac output during exercise, skin perfusion is increased due to profound vasodilation of skin arterioles, reducing cutaneous vascular conductance (cvc) and increasing skin blood flow (skbf). these mechanisms enable an efficient heat transfer to the skin from where it is eliminated and significantly increase the surface area for heat exchange between the body and the environment. the vasodilation of skin microvessels can induce an immense increase of skbf compared to resting conditions, i.e. from 300 ml/min in thermoneutral conditions (while in a cold environment as low as 100 ml/min) up to 7 l/min during exercise in a hot climate (4, 7, 24). the regulation of vascular tone in skin microcirculation is very complex, including neural and local mechanisms (21, 36–39). a decrease in vascular tone is mainly achieved by the withdrawal of the sympathetic tone regulating cvc as the skin arterioles throughout the body are innervated by sympathetic vasoconstrictor nerve fibers (11, 21, 36, 39–41). in addition, non-glabrous non-acral parts of the skin also receive vasodilatory sympathetic fibers which mediate active vasodilation (21, 36, 39). it is speculated that during exercise, these fibers contribute to active vasodilation in non-acral parts of the skin, such as the trunk, skin of the forehead and face, and non-acral parts of extremities (11, 21, 36). while vasoconstrictor fibers mainly mediate their action via noradrenaline acting on α-adrenergic receptors, the exact neurotransmitter of the sympathetic vasodilatory fibers remains questionable: proposed potential (co)transmitters include acetylcholine (ach), substance p, calcitonin-gene-related polypeptide (cgrp) and nitric oxide (no) (21, 36, 38). respectively, the active role of endothelium seems to importantly contribute to active vasodilation in the skin (38, 39, 42). special anatomical features present in skin microcirculation of glabrous acral parts, such as fingers, toes, palms, feet, ear lobes and nose, are arteriovenous anastomoses, direct connections between arterioles and venules (43, 44) which when open significantly increase the rate of heat elimination (24, 36, 43, 45). seemedj 2023, vol 7, no 1 thermoregulation and exercise 17 southeastern european medical journal, 2023; 7(1) figure 1. thermoregulatory mechanisms during exercise. the thermoregulatory center integrates thermal (from central thermoreceptors sensing changes in core temperature, and peripheral thermoreceptors sensing changes in skin temperature (t)) and non-thermal input, compares the temperature perturbation with the “set-point” value, and activates appropriate reflexes to dissipate excessive heat from the body surface by adjusting the response of cutaneous vascular conductance and sweating, respectively. temperature gradient determines the quantitative heat transfer from the core to the surface, where heat is dissipated by four physical principles, whose percentages are variable ( %) and modified by physiological response according to the quantity of the produced heat and ambient conditions, respectively. seemedj 2023, vol 7, no 1 thermoregulation and exercise 18 southeastern european medical journal, 2023; 7(1) evaporation-related heat loss during exercise is increased by sweating another very efficient mechanism of heat elimination is active sweating which is tightly linked to changes in cvc. each gram of water evaporated from the skin surface removes about 2.5 kj of heat from the body (1, 5). sweat glands are innervated by the sympathetic nerve fibers with ach being a putative transmitter acting concomitantly with other cotransmitters (11, 46); an important role of various aquaporins in the skin has recently been reviewed (47). when fully active, sweat glands can increase their sweating rate up to 30 g sweat/min in response to the body’s needs which could account for up to 2-4 l/h during high-intensity exercise in a hot environment (1, 11, 15, 24). indeed, in a hot environment when the ambient temperature exceeds the core temperature, there is no or even a negative temperature gradient for conductive-convective heat transfer, making evaporation the only way to dissipate heat (1, 2, 4, 24). yet, besides physiologic adjustments in the sweating rate and composition of sweat (46, 48–50), the efficiency of sweating also depends on environmental factors, predominantly on the humidity (3, 15, 50, 51). increased humidity can make sweating ineffective when there is no water vapor gradient between the environment and the surface of the body, predisposing to the development of hyperthermia which can be fatal (1, 2). increased skbf and sweating rate persist also far in the recovery period after exercise cessation, depending on the duration and intensity of exercise and the production of heat, until all additional heat is eliminated and the resting temperature steady state is achieved (16, 45, 52–54). a schematic representation of the mechanisms governing thermoregulation during exercise is depicted in figure 1. how could skin blood flow and sweating be assessed the dynamics of skin microcirculation and skin temperature changes during exercise could be traced by using laser doppler fluxmetry (ldf) (39, 45) and the corresponding skin temperature measurement (fig. 2), various laser doppler imaging techniques (55, 56) or ultrasound doppler flowmetry (udf) (57), and have been considered to be a more reliable measure of skbf than plethysmography (58, 59). to obtain a better insight into the particular physiological mechanism behind it, different approaches and algorithms are being developed for the spectral decomposition of the ldf signal (60). interestingly, glabrous and non-glabrous parts of the skin behave differently during exercise and its recovery (45, 57, 61) (fig. 2). a strong cross-coupling between glabrous skbf and core temperature in thermoregulatory function has recently been established based on experimental and modeling data (62). a promising device for tracing sweat rate during exercise as accurately as possible and providing a low-cost device platform to detect other health-relevant biomarkers in the sweat (vapor) as the next-generation sweat sensor for smart healthcare and personalized medicine has been introduced (63). seemedj 2023, vol 7, no 1 thermoregulation and exercise 19 southeastern european medical journal, 2023; 7(1) figure 2. the responses of skin blood flow and the corresponding skin temperature to exercise and its recovery differ between glabrous and non-glabrous areas. a representative tracing of the laser doppler flux (ldf), expressed in arbitrary perfusion units (au) and the corresponding skin temperature (t) in skin microcirculation on the volar forearm (glabrous site) and the finger pulp (non-glabrous site) during graded dynamic exercise and its recovery is shown. exercise load is expressed in watts (w). adapted from (45). the main differences between thermoregulation during rest and exercise: a summary apart from thermoreflexes, many opposing nonthermoregulatory reflexes (mechano-, metabo and baroreflex to list some) are activated during exercise and its recovery to ensure sufficient perfusion of skeletal muscles and the preservation of blood pressure (7, 15, 20, 54, 64– 66). therefore, thermoregulation during exercise importantly differs from that at rest. the most manifest differences are depicted in fig. 3, representing the relation between the body core temperature and the skbf. to assess the characteristics of this relation, i.e. the slope of the curve, the body core temperature and the skbf, potentially with the corresponding skin temperature, should be traced. core temperature is usually determined by an esophageal thermo-sensor, although other methodological aspects have been addressed (5, 20, 22, 62). nevertheless, the data should be interpreted cautiously, especially when measuring peripheral skin t to estimate core temperature, which is rather questionable (67). on the other hand, there are much more options for assessing skin temperature; besides the above mentioned, infrared thermography has gained increasing importance; yet, similarly to other methods, it has not been standardized (68, 69). at the beginning of exercise, an increase of the sympathetic tone throughout the body, presumably due to a central command, enables redistribution of blood flow, increasing cvc and reducing skbf and thus shifting the starting point toward lower skbf at unchanged core temperature (fig. 3) (2, 5, 7, 20, 24). another distinctive difference is a shift in the threshold to induce active vasodilation and sweating in the skin, toward higher core temperatures (fig. 3); interestingly, the threshold shift depends on seemedj 2023, vol 7, no 1 thermoregulation and exercise 20 southeastern european medical journal, 2023; 7(1) exercise intensity and is even more pronounced in high-intensity exercise (11, 20, 36). the dependence of the thermoregulatory threshold on the intensity of exercise might partly explain rapid fatigue and exhaustion during intense exercise due to a higher increase in core temperature, which might even lead to hyperthermia (15, 22, 29). the third pronounced difference is the responsiveness of the effectors in the skin: the maximal vasodilation in the skin during exercise comprises only 60% of the maximal vasodilation attained in resting conditions (fig. 3) (7, 20, 24). pooling of additional blood in the periphery impedes venous return to the heart, and activates baroreflex, leading to “cardiovascular drift” and heart rate increase that is disproportional to the needs of the skeletal muscles (5, 52, 70). figure 3. the main differences between thermoregulation during rest and dynamic exercise. skin blood flow (skbf) in relation to body core temperature is shown. the “starting point” is shifted toward lower skbf at the onset of exercise due to vasoconstriction in the skin (a). the temperature threshold to induce vasodilation is shifted toward a higher core temperature during exercise (b). maximal vasodilation in the skin is reduced compared to resting conditions (c). adapted from (4). in addition, it has been proposed that skin temperature, independently of the core temperature, affects sports performance: maximal oxygen uptake (vo2max) at the same core temperature was shown to be lower at higher skin temperatures than at lower ones (23, 70). increased skin temperature during exercise reduces the temperature gradient for heat elimination, impeding heat exchange. to overcome the decreased temperature gradient, skbf is increased on account of diminished blood flow through skeletal muscles. from the above observations on opposing thermoand non-thermoregulatory reflexes during exercise and its recovery, it is obvious that hyperthermia is more often an issue when exercising than when resting. interestingly, simplified thermoregulation models of the human body exercising in warm conditions have been proposed considering all the above-mentioned players (62, 71). exercise and thermoregulation in extreme environmental conditions hot environment exercising in a hot environment undoubtedly compromises thermoregulation. pooling of additional blood in the periphery increases cardiovascular drift in a hot climate. it has been shown that a heart rate increase of 20 beats/min could not compensate for the decreased stroke volume due to the pooling of blood in the skin: cardiac output was about 1.5 l/min lower during heavy exercise in the heat than during comparable exercise in a cool environment (1, 5, 15). due to insufficient muscle perfusion, the amount of anaerobic metabolism increases, decreasing plasma ph and additionally compromising performance (15, 72). in addition to the pooling of blood, another problem during prolonged exercise is the loss of water and electrolytes, additionally compromising venous return, and consequently jeopardizing the maintenance of blood pressure. to overcome the threatening hypotension, relative cutaneous vasoconstriction issues, rendering the person more susceptible to developing hyperthermia (1, 5, 24). in addition, the reflex of sweating is reset toward higher core temperatures as water preservation due to seemedj 2023, vol 7, no 1 thermoregulation and exercise 21 southeastern european medical journal, 2023; 7(1) reduced osmolarity surpasses the need to eliminate heat by sweating (20, 73–75). respectively, a vicious cycle could ensue leading to hyperthermia and/or shock (35). in this respect, sufficient fluid and electrolytes substitution is a prerequisite for sustaining exercise (24, 34, 76). it has been shown that even 1% of body mass reduction due to dehydration strongly impacts sports performance and vo2max (77). figure 4. the effect of regular training and acclimatization on thermoregulation training and staying in a warm environment for a longer period induce a shift in the temperature threshold for heat elimination (presented as sweating rate) as well as the sensitivity of thermoreflex. adapted from (81). when regularly exercising in a warm environment, acclimatization processes take place which improve sports performance: the sensitivity of thermoreflex is reset toward a lower core temperature threshold (fig. 4), the maximal sweat rate increases and sweat composition changes, becoming more diluted thus helping to preserve body electrolytes (24, 50, 51, 78, 79). in addition, an increase in plasma volume in a longer-term improves circulation and heat exchange (24, 77). all these adaptations improve vo2max and sports performance. interestingly, similar adaptations have also been shown in endurance elite sportsmen exercising in a thermoneutral environment (fig. 4) (48, 73, 78–81). cold environment on the other hand, exercising in a cold environment is not problematic from the thermoregulatory point of view provided it is not of too long duration or performed in cold water which has a 25-fold higher heat conductivity compared to air (5, 25–27, 82). as long as the body core temperature is maintained in a physiological range, the low ambient temperature does not impact sports performance and vo2max. yet, cooling of the muscles impacts their functional abilities in terms of decreasing their strength, maximal force, velocity and reaction time (83, 84). due to vasoconstriction in the skin, the release of free fatty acids from subcutaneous adipose tissue is reduced, limiting the oxidation of fat as fuel for muscle contraction and accordingly increasing the oxidation of carbohydrates. potential hypothermia results from a drop in core temperature when exercising for a longer time, or insufficiently dressed or performing water sports (26). acclimatization to cold induces adaptive mechanisms which enable better preservation of heat on one side and increased production on the other, including shivering and brown fat adipose tissue thermogenesis (2, 25–27). effect of sex and age on thermoregulation during exercise an interesting issue to be discussed is the effect of sex on thermoregulation. on one side, centrally mediated mechanisms might affect the response regarding sex, and on the other, sex hormones are known to exert direct peripheral effects on the vascular tone (11, 18, 19, 36, 85). yet, studies reported controversial results. most often, the main differences are attributed to the sweating response: the slope of the curve representing sweating rate dependency on the body core temperature was reduced in women compared to men (86, 87) even though women exhibited a greater number of sweat glands (51). the differences in the sweat rate were more pronounced when exercising at higher intensities (86). it seems that the temperature threshold to induce vasodilation in the skin is seemedj 2023, vol 7, no 1 thermoregulation and exercise 22 southeastern european medical journal, 2023; 7(1) higher in women (85). however, a recent metaanalysis has shown that despite increased core t in women in the luteal phase of the menstrual cycle this does not significantly impact thermoregulatory response, at least in terms of sweat rate and skin temperature (19, 88). finally, it is worth emphasizing that aging also affects the thermoregulatory response to exercise (1). effect of endurance training on thermoregulation during exercise regular training in both men and women induces significant adaptations of the thermoregulatory system, including cutaneous vascular reactivity as well as sweating. the threshold to activate effectors for heat elimination is lower in trained compared to sedentary (64, 77, 81, 89) (fig. 4). sweat rate and composition are altered in terms of increased sweat rate at the same exercise intensity (expressed as % vo2max) and more diluted sweat, respectively enabling more efficient electrolytes preservation in sportsmen (51, 78, 80). in addition, sportsmen have been shown to better acclimatize to the heat, which rather than reflecting structural changes has been attributed to functional vessel alterations (79, 90). a part of altered vascular responsiveness might be attributed to increased endotheliumdependent vasodilation (91, 92) which might additionally contribute to beneficial thermoregulatory adaptations. all these adaptations enable sportsmen better adjustments to physical and thermal stress, reaching higher vo2max values and performing exercise for longer periods, in thermoneutral, as well as in extreme environmental conditions. conclusion thermoregulation during exercise differs from that in resting conditions since, during exercise, thermoregulatory reflexes oppose the nonthermoregulatory ones. the opposing reflexes integrate at the level of skin microcirculation which is the main organ for heat elimination. an important issue when exercising is a sufficient substitution of water and electrolytes, especially in a warm and humid environment. regular physical activity induces several beneficial changes, including better thermoregulatory mechanisms. yet, exact mechanisms, as well as the most optimal training regime, remain to be determined, and above others, appropriate methods to measure the many players involved in thermoregulation in humans in vivo need to be established. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. mcardle wd, katch fi, katch vl. essentials of exercise physiology. 3rd ed. mcardle wd, katch fi, katch vl, editors. baltimore, philadelphia: lippincott williams & wilkins; 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6(2) original article do water molecules displaced by hydrophobic interactions stabilize antigen-antibody binding?1physico-chemical background of antigen-antibody reactions analyzed by fluorescent and fourier-transform infrared spectroscopy on fitc – anti-fitc (igg1) model peter németh* department of immunology and biotechnology, clinical centre, university of pécs medical school, pécs, hungary *corresponding author: peter németh, nemeth.peter@pte.hu received: oct 12, 2022; revised version accepted: oct 27 2022; published: nov 28, 2022 keywords: antigen-antibody reaction, fitc, fluorescence, spectrum analysis abstract background: antigen-antibody reactions are a special field of molecular interactions. the physicochemical nature of antigen-antibody binding and ligand-induced changes in the fine molecular structures of antigens during immunocomplex formation are less studied. however, these changes in the molecular appearance are extremely important for further molecular recognition. the major aim of this study is to clarify the physico-chemical modification of the antigen/hapten during immunobinding using model experiments. methods: an appropriate model system was designed for our investigations: fluorescein-isothiocyanate (fitc, isomer i) was used as the antigen (hapten), and its interactions with a specific antibody (monoclonal anti-fitc igg1) were analyzed using spectrophotometry, different spectrofluorimetric methods and fluorescence polarization, and fourier-transform infrared spectroscopic methods. results: fluorescent polarization and infrared spectroscopic measurements detected a local decrease in the hydration degree in the submolecular area of the specific ligand between the small antigen (hapten) molecule and the hypervariable region of the specific igg1, causing “rigidization” of molecular movements. changes in hydration modified the molecular microenvironment, allowing them to influence further functions of both immunoglobulins and the antigen. conclusion: hydrophobic interactions with exclusion of water molecules around the binding sites seem to be thermodynamically strong enough for stable molecular binding without a covalent chemical interaction between the antigen and the antibody. the results of this study, together with data obtained in previous research, help understand the molecular dynamics of the antigen-antibody reaction better. (németh p. do water molecules displaced by hydrophobic interactions stabilize antigen-antibody binding?. seemedj 2022; 6(2); 1-19) seemedj 2022, vol 6, no. 2 physico-chemical background of antigen-antibody reactions 2 southeastern european medical journal, 2022; 6(2) introduction antigen-antibody interactions are special forms of molecular associations in vivo and in vitro. the influence of antigen molecules on specific immunoglobulins during the formation of immune complexes has been studied: modification of the physico-chemical character of immunoglobulins after the binding of an antigen can influence the biological functions of an antibody (e.g. complement fixation, fc receptor functions, etc.) (1, 2, 3, 4). however, our knowledge about diverse modifications of the fine molecular structure of antigens, especially changes in the structure of low molecular weight antigens (or so-called haptens) by specific immunoglobulins, is limited (5, 6, 7). however, these physico-chemical changes in the fine structure of antigens cross-linked by immunoglobulins seem to be important in the further regulation of several other molecular interactions. activation or inhibition of different receptors, enzymes, and transporter molecules interchange during immunocomplex formation. both new physiological and pathological immune reactions occur by the modification of the primary physico-chemical appearance of the antigenic structure after binding with an immunoglobulin (8). the submolecular mechanism of immunobinding and consequential modifications of fine molecular structures are poorly understood (9, 10, 11). simple electrostatic coherence between the antigen and immunoglobulins is not thermodynamically strong enough to form a stable molecular complex and cause remarkable modifications of physico-chemical appearances of participating molecules (12, 13). an analysis of hapten-immunoglobulin interactions is more favourable for studying the physico-chemical nature of structural modifications of the antigen caused by immunobinding. we have developed an experimental model system that allows us to use spectrophotometry, spectrofluorimetry, different fluorescence polarization techniques, and fourier-transform infrared spectroscopy (ft-ir) as delicate methods for a complex analysis of the physico-chemical nature of hapten-immunoglobulin interactions. a conventional fluorescent dye (fitc) was used as the antigen and a specific monoclonal antibody (anti-fitc igg1) against this hapten molecule had been developed at our department earlier (14). immunological recognition and the consequential physico-chemical changes in the antigen/hapten structure were studied by carrying out a comparative analysis of native and chemically cross-linked fitc molecules. the further aim of this study was to find theoretical explanations for the feature of strong molecular interactions between the antigen and antibody, which are thermodynamically equivalent to a covalent chemical bond, without a real chemical ligand formation (15). materials and methods antigen: fluorescein-iso-thiocyanate (fitc, isomer i) was used as a hapten (mw: 389.4, product of the sigma chemical company, usa), and its interactions with a specific antibody were studied using spectrophotometry, different spectrofluorimetric methods, fluorescence polarization, and fourier-transform infrared spectroscopic methods. for the preparation of the standard antigen stock solution, 1 mg fitc was dissolved in 100 µl dimethyl-sulfoxide (dmso, sigma chemical company, usa), then diluted to 1 mg/ml concentration with pbs 0.15 m, ph 7.2 (phosphate buffered saline containing 5.4 mm na2hpo4, 1.5 mm kh2po4, 140 mm nacl and 2.7 mm kcl). this stock solution was kept at 4 °c. for the comparative analysis of free (native) fitc and the carrier cross-linked form, different protein molecules were labelled with fitc as bovine serum albumin (bsa, sigma chemical company, usa), polyclonal anti-ratigg antibody (produced in sheep by our laboratory), and monoclonal mouse antibodies with igg1 isotype (anti-fitc, anti-βhcg, antiinsulin produced by our department earlier). the fitc-labelling of proteins was carried out by following the method described (17). after protein labelling, the free, non-conjugated fitc seemedj 2022, vol 6, no. 2 physico-chemical background of antigen-antibody reactions 3 southeastern european medical journal, 2022; 6(2) was removed from the reaction mixture by gelfiltration on a sephadex g 25 fplc column (pharmacia, sweden) equilibrated with pbs. afterwards, the so-called conjugation rate – that is, the number of fitc molecules coupled onto one protein molecule – was determined semiquantitatively by measuring the optical density of solutions of labelled proteins at 495 and 280 nm and by applying an empirical formula for the calculation: x a a a = − 2 87 0 35 495 280 495 . . * * where x is the conjugation rate and a280 and a495 are the optical density of the solution measured at 280 and 495 nm, respectively (18). the conjugation rate was 10.7 for fitc-bsa, 8.6 for fitc-anti-rat-(sheep) igg, 12.4 for anti-fitc igg1, 10.3 anti-βhcg, and 11.3 for anti-insulin igg1 antibodies in our experiments. antibody: the monoclonal anti-fitc antibody (mouse, igg1) was developed at our department earlier (14). the mass of the antibody was produced by hybridoma fermentation using harvestmouse (serotec, uk) hollow-fibre fermenter and affinity-purified on a fitc-bsa-coupled sepharose 4b column. the column was prepared as usual (16). in brief, 1.8 g of cnbractivated sepharose 4b (pharmacia, sweden) gel was suspended, swollen and washed five consecutive times in 50 ml 1 mm hcl. 100 mg bsa (sigma chemical company, usa) was dissolved in 10 ml coupling buffer, that is 0.1 m nahco3, ph 8.3, containing 0.5 m nacl, and mixed with the gel. the mixture was being rotated end-over-end for two hours at room temperature. following a thorough wash in the coupling buffer, the gel was left to react with 300 µg/ml fitc, generally following the fitclabelling method described above (17). afterwards, the reaction was stopped and all remaining active groups were blocked by washing in 50 ml 0.1 m ph 8 tris-hcl buffer for two hours at room temperature. the gel was then washed in five cycles of alternating ph. each cycle consisted of a wash with 0.1 m ph 8 acetate buffer containing 0.5 m nacl. following this washing procedure, the gel – adding up to 7 ml – was poured into a column and equilibrated with pbs containing 0.05% nan3. the anti-fitc antibody was purified on the aforementioned column, thus ensuring that all antibody molecules would recognize the hapten selectively. 800 ml hybridoma supernatant was flown slowly through the column for 24 hours at 4 °c. the antibody was then eluted from the column with 0.1 m ph 2.5 glycine buffer. 1-ml fractions were collected and 100 µl 1 m ph 9 tris-hcl was added instantly to each of them. afterwards, the concentration was determined by measuring the optical density at 280 nm. the purified monoclonal anti-fitc antibody was fragmented to fab using papain as usual (19). the purified anti-fitc antibody was digested with papain in the presence of a reducing agent, cysteine. to determine optimal conditions, pilot fragmentation was carried out, and both the concentration of papain and the time of digestion were varied. after the pilot fragmentation, the fragments were dialyzed and analyzed by phastsystem sds-page fast gel electrophoresis (pharmacia-lkb, sweden). optimal conditions were determined according to the gel results. these conditions were used in the large-scale fragmentation of anti-fitc to anti-fitc fab. 5 ml anti-fitc igg1 monoclonal antibody (with a concentration of 2 mg/ml) was added to a freshly-mixed digestion buffer (pbs 0.15 m, ph 7.2, 0.02 m edta, 0.02 m cysteine), containing 0.1 mg/ml papain. the enzymeantibody ratio was 1:20. the reaction mixture was well-mixed and incubated in a water bath at 37 °c. after six hours, the mixture was removed from the water bath, and 1 ml of 0.3 m iodoacetamide in pbs was added to stop the reaction. the reaction mixture was dialyzed against 2 litres pbs, ph 8.0, for 24 hours at 4 °c. the products were analyzed with pharmacia’s phastsystem sds-page fast electrophoresis. a 10 x 200 mm protein a-sepharose cl-4b (pharmacia, sweden) column was made, and the seemedj 2022, vol 6, no. 2 physico-chemical background of antigen-antibody reactions 4 southeastern european medical journal, 2022; 6(2) dialyzed reaction mixture was loaded onto it. unbound fractions containing the fab fragment and enzyme were collected and the column was washed with pbs to remove the fab fragments completely. the mixture was concentrated to 5 ml. a 26 x 900 mm sephacryl s-200 superfine column (pharmacia, sweden) was made and the concentrated reaction mixture was loaded onto it. fractions of 50 kd were collected and the purity of the final product was checked using 10% nonreducing sds-polyacrylamide gel (pharmacia’s phastsystem). the concentration of the fab fragments was assessed at a280 and stored in borate buffer (0.015 m sodium borate, 0.15 m nacl, ph 8.5) at 4 °c. we used monoclonal anti-βhcg antibody (mouse, igg1) as an isotype (negative) control, which had also been developed at our department earlier. the production, purification, labelling, and preparation of the fab fragments were carried out as described in case of the antifitc igg1 monoclonal antibody. spectrophotoand fluorimetry, ft-ir spectroscopy: in order to avoid the so-called “inner filter effect” (this phenomenon can be seen when a solution of a given compound is concentrated enough to absorb an already significant ratio of the exciting light beam, thus reducing light intensity at the centre of the cuvette where the emitted light is detected, consequently interfering with the results), prior to fluorimetric measurements, all samples were diluted in pbs 0.15 m ph 7.2 to a concentration at which the optical density of the solution measured at its absorption and emission peaks was below 0.05. this concentration was 50 ng/ml for free fitc, 5 µg/ml for fitc-bsa, 10 µg/ml for fitc-labelled anti-rat-igg, and 10 µg/ml for monoclonal antibodies with igg1 isotype. non-diluted protein concentrations were determined by measuring the optical density at 280 nm for all immunoglobulins and by using the dye-binding assay for bsa as described (20, 21). spectrophotometric measurements were performed on a uv/vis photometer (du68, beckman instruments inc., usa) using 2 x 10 mm quartz cuvettes, all at room temperature. some fluorescence-quenching measurements were done on a steady-state fluorimeter (locarte, england), in cylindrical cuvettes of 3 mm in diameter, at room temperature. a 330 w zn arc lamp was used as the excitation light source. 490 nm excitation wavelength was selected using the lf3 monochromatic filter. emission was monitored through lf4 and lf7 cutoff filters and the 510570 nm range was therefore selected. the majority of steady-state and all spectrofluorimetric measurements were performed on a hitachi-perkin elmer (mpf 4) spectrofluorimeter equipped with polarization filters and a thermostated cell holder in 10 x 10 mm quartz cuvettes operated in ratio mode, all at room temperature. the light source was a 300 w xe arc lamp. both excitation and emission wavelengths were set by monochromators and the precise values were dependent on the actual fluorescent solution. slits were 4 and 8 nm, respectively. fluorescence polarization measurements were achieved on the hitachiperkin elmer (mpf 4) spectrofluorimeter. with each sample, four measurements of fluorescence intensity were taken using one pair of polarization filters. the first sample was taken using vertically polarized exciting light and detecting vertically polarized emitted light (ivv), the second included vertical excitation and horizontal emission (ivh), the third involved horizontal excitation and vertical emission (ihv), and the fourth sample included horizontal excitation and horizontal emission (ihh). following these measurements, the fluorescence anisotropy of fitc or fitclabelled proteins could be calculated using an appropriate equation (see below). fluorescence life span measurements were taken on an iss multifrequency phase fluorimeter (iss fluorescence instrumentation, champaign, illinois, usa) using the frequency cross-correlation method. a 300 w xe arc lamp was used as the excitation light source. excitation light intensity was modulated using a seemedj 2022, vol 6, no. 2 physico-chemical background of antigen-antibody reactions 5 southeastern european medical journal, 2022; 6(2) double-crystal pockel cell and a two-way polariser. a cross-correlation frequency of 80 hz was used. the excitation wavelength was set to 493 nm and the emission was also monitored through a monochromator set to 512 or 522 nm. a freshly prepared glycogen (sigma chemical co., usa) solution was used as a reference to correct the instrumental phase delay of the detection equipment. ten sets of phase and modulation data were collected for each sample. phase and modulation data were analyzed using the iss 187 decay analysis software. fluorescence anisotropy was calculated using the following equation: r vv vv g vh vh vv vv g vh vh s s s s = − − − − + − ( ) ( ) ( ) ( )2 where r is fluorescence anisotropy; vv and vh are the fluorescence intensity measured with polarization filters set vertically in the excitation and vertically or horizontally in the emission light beam, respectively; vvs and vhs are the fluorescence intensity of control (fitc) samples measured with polarization filters set vertically in the excitation and vertically or horizontally in the emission light beam, respectively; and g is a correction factor, which is calculated using the following equation: g hv hv hh hh s s = − − where hh and hv are the fluorescence intensity measured with polarization filters set horizontally in the excitation and horizontally or vertically in the emission light beam, respectively; hhs and hvs are the fluorescence intensity of control (fitc) samples measured with polarization filters set horizontally in the excitation and horizontally or vertically in the emission light beam, respectively. (22, 23) the perrin equation was applied to analyze fluorescence anisotropy of a given fluorophore depending on its molecular microenvironment: 1 1 1 0r r f d = +( )   where r and r0 are anisotropy and so-called limit anisotropy – the anisotropy extrapolated to 0 k temperature; tf and td are fluorescence lifetime and rotation correlation time (22, 23). fourier-transform infrared spectroscopy (ft-ir) measurements were taken on a spectra 400 ftir spectrometer (nicolet, usa). 1-mg samples were lyophilized for 24 hours in a savant speed vac lyophilizer (speed vac plus sc210a, savant, usa). the lyophilized samples were desiccated further for 68 hours in a vacuum desiccator filled with silica crystals. 0.1-0.5 mg of the dehydrated samples were pastillized in kbr. the fouriertransform infrared transmittance spectrum was measured at a range of 400-4000 cm-1 and studied with a 4 cm-1 resolution. during the investigations of samples, the ir spectra of water vapor, carbon dioxide, and water of potassium bromide were subtracted from the total numeric data before the assignation. the ir bands were identified according to data in the relevant literature (24, 25, 26, 27). for the analysis of solvent polarity, acetone was used in different dilutions as a nonpolar solvent. fitc was dissolved in dmso, as usual, but afterwards it was diluted in mixtures of acetone and water in various ratios. these samples were measured in cuvettes covered with a lid to prevent the acetone from evaporating. the fluorescence intensity and light absorbency was detected in a thermostatic fluorimeter at 20 °c. florescent spectroscopic measurements were repeated from two up to ten times independently and the scattering between the results was under 3% in all cases. results the basic finding was that fitc fluorescence intensity definitely suffers from a fluorescencequenching effect following pre-incubation with our anti-fitc igg1 monoclonal antibody (12). similar observations were made in case of using polyclonal and monoclonal (high affinity igm) antibodies against fitc (9, 28, 29). our model permits kinetic studies with different fluorescent seemedj 2022, vol 6, no. 2 physico-chemical background of antigen-antibody reactions 6 southeastern european medical journal, 2022; 6(2) and infrared spectroscopic measurements by the advantageous structure of the mouse igg1 molecule and its fab fragment. during the optimization of fluorescence quenching measurements, fitc with anti-fitc antibody were incubated together for 1 10 minutes at room temperature in order to let the equilibrium be established, and then the fluorescence intensity of the reaction mixture was quantified. a trial to measure the time needed for anti-fitc to fully achieve its fluorescence-quenching effect was performed. we added a suitable amount of anti-fitc to a cuvette with the fitc solution already inside the fluorimeter and tried to monitor the decline of fluorescence intensity – with the results verifying our assumptions, since there was an instant quenching of fluorescence. according to our pilot study, the optimum incubation time for maximum fluorescence quenching caused by the antifitc monoclonal antibody was 10 minutes. no similar fluorescence-quenching effects were found when fitc was preincubated by nonspecific monoclonal antibodies with the same isotypes. we found total (100%) inhibition of fitc fluorescence intensity induced by both antifitc monoclonal antibody and its fab fragment (figure 1). figure 1. fitc fluorescence quenching by anti-fitc monoclonal antibody and its fab fragment. fluorescence intensity presented in relative units of the fluorimeter. excitation and emission wavelengths were set to 493 and 512 nm. measurements were taken at ph 7.2 at 21 °c. the figure demonstrates data of one typical measurement. scattering between five independent assays was under 3%. in the figure, we demonstrated two separate curves of fluorescence intensity – one for 10 μ g/ml anti-fitc antibody and the other for 10 μ g/ml anti-fitc fab – against the concentration of fitc. it is striking that at 15 ng/ml fitc concentration, the fluorescence was quenched by 50% already in the case of anti-fitc fab. the fluorescence was quenched by 50% in case of anti-fitc at around 5 ng/ml fitc concentration. at the equal paratope and hapten ratio (when the absolute number of hapten molecules theoretically equals the binding sites of the antibody molecules) , total (100%) quenching of fluorescence occurred. at a higher hapten concentration – over 200 ng/ml – a slight innerfilter effect can be seen at the beginning of the seemedj 2022, vol 6, no. 2 physico-chemical background of antigen-antibody reactions 7 southeastern european medical journal, 2022; 6(2) curve. at about the threshold concentration of fluorophore (that is, when the optical density of the solution measured at its excitation peak equals 0.05), the partial quenching effect of a few anti-fitc molecules causes the same phenomenon as dilution. in spite of the lesser quantity of fluorescent compound actually emitting, the fluorescence intensity virtually enhances. afterwards, with the addition of more antibody, the fluorescence intensity declines quickly to total quenching of fluorescence. this occurs at different hapten and paratope molecular ratios, depending on their total concentration, which is deductible from the law of mass action. in the following phase of our study, we tried to analyze how this non-covalent molecular interaction – an immune bond – can modify the physico-chemical properties of an antigen molecule to such a fundamental degree. in general, when the fluorescence intensity of a given fluorescent molecule decreases because of interaction with another molecule, the first interpretation that emerges is spectral shift. we investigated this possibility too, but found no remarkable spectral change if fitc molecules were bound chemically onto the surface of different protein carriers (figure 2). figure 2. emission spectrum of fitc covalently bound onto different protein molecules. excitation wavelengths were set to 493 nm for free fitc, 503 nm for fitc-bsa, 496 nm for fitc-igg1. measurements were taken at ph 7.2 at 21 °c. scattering between three independent measurements was under 5%. since covalent binding onto the surface of such big molecules as immunoglobulins or albumin (bsa) did not result in any spectral-shift bigger than 10 nm, it is hard to uphold the assumption that a simple, non-covalent molecular association between the small fluorophore and a relatively big partner molecule (a specific antibody in our model) causes fluorescence quenching. this result suggests that the mechanism of fluorescence quenching caused by anti-fitc monoclonal antibody is different from the covalent chemical binding, because it must be the unique nature of a specific immune bond that causes quenching. to find a better explanation for this phenomenon, we compared the molecular movement and rotational freedom of free fitc 0 20 40 60 80 100 120 500 508 512 516 520 524 540 559 in te n si ty o f e m it te d l ig h t wavelength (nm) fitc 493nm fitc-bsa 503nm fitc-igg 496nm seemedj 2022, vol 6, no. 2 physico-chemical background of antigen-antibody reactions 8 southeastern european medical journal, 2022; 6(2) with fitc molecules covalently bonded to proteins, or with immunologically bonded fitc by anti-fitc or its fab fragment using fluorescence polarization measurements. results of these measurements showed further fundamental differences in the physicochemical nature of covalent (chemical) and noncovalent (immunological) bonds. using this technique, we were able to quantitatively detect the slightest changes in the rotation and/or flexibility of fluorophore molecules if they were limited in any way by any other molecule. the results of our fluorescent polarization measurements are presented in figure 3. figure 3. fluorescence anisotropy of free fitc and fitc bound to proteins chemically or bound by antifitc and anti-fitc fab. excitation wavelengths were set to 493 nm for fitc, 503 nm for fitc-bsa, 496 nm for fitc-igg1 and fitc-fab, while the emission wavelengths were 512 nm for fitc, 522 nm for fitcbsa, and 515 nm for fitc-igg1 and fitc-fab. measurements were taken at ph 7.2 at 21 °c. the figure demonstrates summarized data of five independent measurements. scattering was under 3%. according to the perrin equation, fluorescence anisotropy of a given fluorophore depends on its molecular microenvironment. this equation says that the bigger the anisotropy, the shorter fluorescence lifetime and the longer rotation correlation. in other words, an increase in anisotropy means that a fluorophore can transmit the energy it absorbs to another molecule in a very short time. this occurs because there is a very close association between the two molecules, which in some cases leads to instant channelling of absorbed energy from the fluorophore to the other molecule; moreover, this interaction can almost totally block the rotation of the little fluorescent molecule. based on this equation, we can draw basic conclusions concerning the molecular interactions demonstrated in figure 3. the free movement and rotation of fitc molecules gets partially restricted after conjugation with higher molecular weight carrier proteins, such as bsa, or an indifferent, mouse igg, characterized by increased anisotropy. however, the molecular movement of fitc is definitely inhibited after the incubation with specific anti-fitc monoclonal antibody. immunological binding has a much more dramatic effect on fitc rotation and energy transfer than covalent chemical binding to indifferent proteins, including immunoglobulins with the same isotype (igg1) the molecules have. the anisotropy was 100% higher in case of immunocomplexes formed by seemedj 2022, vol 6, no. 2 physico-chemical background of antigen-antibody reactions 9 southeastern european medical journal, 2022; 6(2) fitc and anti-fitc (or anti-fitc fab) than in case of covalent binding to the same isotype (igg1) the molecule has. anisotropy difference of only 5% was measured between the whole anti-fitc igg1 and their fab fragment during the formation of immunocomplexes with free fitc. another proof of a specific molecular movement-inhibiting, “rigidizing” effect of immune binding compared to that of covalent, chemical binding came from fluorescence lifetime measurements. these data are demonstrated in figure 4. figure 4. fluorescence lifetime of free fitc and fitc bound to bsa, bound by anti-fitc igg1 and antifitc fab. excitation wavelengths were set to 493 nm for fitc, 503 nm for fitc-bsa, 496 nm for fitcigg1 and fitc-fab. measurements were taken at ph 7.2 at 21 c. the figure demonstrates summarized data of five independent measurement. scattering was under 3%. fluorescence lifetime gives us valuable information on the milieu surrounding the fluorophore molecule. the shorter the life spans, the quicker the energy transfer between the fluorescent molecule and its environment – that is, the interaction between the two molecules is stronger. it is very well demonstrated that lifetime is greatly affected by the milieu: fitc in pbs has a lifetime of about 4 ns, while bsacoupled or igg1-coupled fitc has a bit shorter lifetime, but on the same scale. we practically cannot measure the lifetime of fitc in fitc– anti-fitc (or fitc–anti-fitc fab) complexes, which is not surprising, since there is practically no fluorescence signal because of quenching. the lifetime of fitc measured was about 4 ns when there was free unbound fitc in the reaction mixture. however, when anti-fitc immunoglobulins or their fab fragments consumed all free fitc molecules, together they formed a so-called “dark complex”. this meant there was no measurable fluorescence signal, so we could determine that lifetime equalled zero. the “rigidization” of fitc molecules by immunobinding to anti-fitc igg1, characterized by increased anisotropy and a shorter fluorescence lifetime, is a really striking phenomenon. however, this “high affinity interaction and energy transfer” theory simply cannot explain another astonishing finding that light absorption of fitc is similarly affected – inhibited – by anti-fitc as its fluorescence intensity. this is presented in figure 5. 0 0.5 1 1.5 2 2.5 3 3.5 4 fitc fitc-bsa fitc + antifitc fitc + antifitc fab f lu o r e sc e n c e l if e ti m e (n s) seemedj 2022, vol 6, no. 2 physico-chemical background of antigen-antibody reactions 10 southeastern european medical journal, 2022; 6(2) figure 5. light absorption of immunocomplexes formed by anti-fitc igg1 and anti-fitc fab plotted against the concentration of fitc. excitation wavelengths were set to 493 nm. measurements were taken at ph 7.2 at 21 c. scattering between two independent measurements was under 2%. based on these data, it can be said that both fluorescence intensity and light absorption are greatly decreased by anti-fitc igg1 or its fab fragment. while the energy transfer from the excited fluorophore molecule can really be responsible for the decreased fluorescence intensity, it cannot explain light absorption at all. no remarkable differences were found between the whole immunoglobulin igg1 (molecular weight is approximately 150 kd) and its fab fragment (approximate molecular weight is 45 kd). we did not find any spectral shift caused by anti-fitc monoclonal igg1 antibody or fab fragments. the difference between polar and nonpolar microenvironment was analyzed. figure 6 demonstrates the effect of acetone as a nonpolar solvent on fitc fluorescence. seemedj 2022, vol 6, no. 2 physico-chemical background of antigen-antibody reactions 11 southeastern european medical journal, 2022; 6(2) figure 6. fluorescence intensity of fitc dissolved in mixtures of acetone and water plotted against the concentration of acetone. excitation and emission wavelengths were set to 493 and 512 nm, respectively. fluorescence intensity presented in relative units of the fluorimeter. measurements were taken at ph 7.2 at 21 c. the figure shows a typical curve. scattering between three parallel measurements was under 2%. the data clearly show that acetone has a similar decreasing effect on fitc fluorescence as immunobinding by anti-fitc igg1 monoclonal antibody. figure 7. light absorption of fitc in mixtures of acetone and pbs measured at 493 nm plotted against the concentration of acetone. measurements were taken at ph 7.2 at 21 c. the figure shows a typical curve. scattering between three parallel measurements was under 2%. 0 500 1000 1500 2000 2500 3000 0 20 40 60 80 100 f lu o r e sc e n c e i n te n si ty acetone concentration (% v/v) 100 ng/ml fitc 0 0.3 0.6 0.9 1.2 1.5 1.8 2.1 0 20 40 60 80 100 o d 4 9 3 acetone concentration (% v/v) 10 µg/ml fitc seemedj 2022, vol 6, no. 2 physico-chemical background of antigen-antibody reactions 12 southeastern european medical journal, 2022; 6(2) table i. spectrum assignation fitc position intensity assignment 682,303 56,020 c=c 728,317 72,842 =ch 761,780 67,621 =ch 855,877 43,726 =ch 1462,625 33,307 ccar 1487,071 54,354 ccar 1534,027 35,647 ccar 1596,729 13,929 ccar 1642,077 65,418 co 2033,843 36,576 asn=c=s 3052,083 80,938 =ch 3071,663 80,675 =ch anti-fitc (igg1) fitc immunocomplex position intensity assignment 1332,642 78,635 amid iii 1410,816 65,668 s co2-, zwitter-ion 1441,279 81,034 c=car* 1508,936 73,703 amid ii, s nh3+ 1632,279 41,895 amid i, as nh3+, nh2 flodsheet 2037,691 86,476 nh3+ 2964,388 79,044 aliphatic ch 3069,372 71,81 oh, humidity 3426,762 62,718 oh, humidity anti-fitc (igg1) position intensity assignment 1333,148 76,502 amid iii 1411,488 62,640 s co2-, zwitter-ion 1508,429 69,217 amid ii, s nh3+ 1632,578 44,031 amid i, as nh3+, nh2 flodsheet 2037,599 84,468 nh3+ 2964,737 71,120 aliphatic ch 3090,425 62,214 oh, humidity 3236,178 68,515 oh, humidity anti fitc (igg1) fitc covalent binding (fitc labbeled igg1) position intensity assignment 1289,048 92,069 amid iii 1411,747 86,795 s co2-, zwitter-ion 1459,746 86,069 c=car* 1510,120 83,004 amid ii, s nh3+ 1534,883 83,579 c=car* 1648,909 82,896 amid i, as nh3+, nh2, disorganized protein sequences 1656,884 82,505 amid i, as nh3+, nh2 helix, co* 2036,716 89,831 nh3+ 2933,257 96,469 aliphatic ch 3355,729 93,908 oh, oh, humidity *fitc seemedj 2022, vol 6, no. 2 physico-chemical background of antigen-antibody reactions 13 southeastern european medical journal, 2022; 6(2) fluorescence quenching increases proportionally to the concentration of acetone and total (100 %) quenching of fluorescence occurs at 96 % acetone concentration (v/v), when fitc molecules practically have no hydrate wrap. moreover, the same phenomenon can be observed with respect to light absorption (figure 7). infrared spectroscopy can analyze more precisely the molecular connections between the antigen and antibody structures, including the influence of the microenvironment. according to our fourier-transform infrared (ftir) spectroscopic measurements in case of covalent binding peak 2033 cm-1 of fitc’s n-cs group (which is the covalent binding region) showed remarkable changes; it disappeared. only the peak 2036 cm-1 of anti-fitc’s nh3+ groups was present. figure 8 demonstrates the ft-ir spectrum of free fitc. univ.med.school fitc tue jun 15 14:36:09 1999 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 % t r a n s m i t t a n c e 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 wavenumbers (cm-1) figure 8. fourier-transform infrared spectra of free fitc molecules. the 0.2 mg sample was pastillized in kbr at a range of 400-4000 cm-1 and studied with a 4 cm-1 resolution. the details of peak assignations are presented in table i. figure 9 shows the spectrum of free anti-fitc igg1, and figure 10 shows the spectrum of fitc covalently bound to monoclonal anti-fitc igg1. seemedj 2022, vol 6, no. 2 physico-chemical background of antigen-antibody reactions 14 southeastern european medical journal, 2022; 6(2) univ.med.school ig-g1 fri jun 25 15:27:23 1999 45 50 55 60 65 70 75 80 85 90 95 100 % t r a n s m i t t a n c e 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 wavenumbers (cm-1) figure 9. fourier-transform infrared spectra of anti-fitc igg1 monoclonal antibody molecules. the 0.5 mg sample was pastillized in kbr at a range of 400-4000 cm-1 and studied with a 4 cm-1 resolution. the details of peak assignations are presented in table i. univ.med.school igg fitc labelled (covalently) fri jun 25 15:51:15 1999 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 % t r a n s m i t t a n c e 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 wavenumbers (cm-1) figure 10. fourier-transform infrared spectra of igg1 monoclonal antibody covalently labelled by fitc. the 0.5 mg sample was pastillized in kbr at a range of 400-4000 cm-1 and studied with a 4 cm-1 resolution. the details of peak assignations are presented in table i. major characteristics of native fitc and igg1 molecules can be found in the spectrum of the complex. seemedj 2022, vol 6, no. 2 physico-chemical background of antigen-antibody reactions 15 southeastern european medical journal, 2022; 6(2) specific binding sites of the anti-fitc igg1 were blocked before and during the covalent fitclabelling. the spectrum contains characteristic peaks of both fitc and anti-fitc igg1. the peaks of poly-aromatic cyclical structures (responsible for fluorescence of fitc molecules) added to the peaks of the anti-fitc igg1 molecule: peaks 1459 cm-1 and 1534 cm-1 were clearly shown and peak 1642 cm-1 shifted and added to anti-fitc’s 1656 cm-1 peak. in case of covalent binding, we did not find hydrophobic interactions between the antigen and antibody molecules: the anti-fitc igg1’s 3236 cm-1 oh peak shifted to 3355 cm-1. in case of immunobinding between the fitc and anti-fitc igg1, the antigen’s n-c-s group was assigned to peak 2033 cm-1, that is, the covalent binding region of fitc. the peak 2033 cm-1 showed no remarkable changes during antigen-antibody reactions, but only shifted and added to the peak 2037 cm-1 of nh3+ groups on antibodies. the polyaromatic cyclical structure, which is responsible for fluorescence, showed significant physico-chemical changes: peaks from 1462 cm-1 to 1642 cm-1 disappeared and/or shifted to 1441 cm-1. however, hydrophobic interactions were detected between the antigen and antibody molecules: the antibody’s 3090 cm-1 and 3236 cm-1 peaks of oh groups shifted to 3069 cm-1 and 3426 cm-1, and their transmittance decreased (figure 11). univ.med.school igg + fitc complex (1/2) fri jun 25 15:42:47 1999 45 50 55 60 65 70 75 80 85 90 95 100 % t r a n s m i t t a n c e 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 wavenumbers (cm-1) figure 11. fourier-transform infrared spectra of immunocomplexes formed by fitc and anti-fitc igg1. the 0.2 mg sample was pastillized in kbr at a range of 400-4000 cm-1 and studied with a 4 cm-1 resolution. the details of peak assignations are presented in table i. no polyaromatic peaks of fitc were found (1400-1600 cm-1) and peaks of oh groups of free anti-fitc igg1 (3000-3400 cm-1) changed remarkably in the immunocomplex. discussion our conclusion is that immunobinding itself can modify the electron cloud of antigens so dramatically that they could not exhibit the same electrostatic structures anymore. we concluded that this extreme structural modification results from making it impossible for the fitc molecule to get in contact with water. it has been known for a long time that solvents have a basic influence on the fluorescence of fluorescent compounds (30, 31). considering this, we suspected that this phenomenon is caused by seemedj 2022, vol 6, no. 2 physico-chemical background of antigen-antibody reactions 16 southeastern european medical journal, 2022; 6(2) the lack of water in the microenvironment of fitc bound by anti-fitc igg1. we tried to model water exclusion by using non-polar solvents for making our samples. the spectral shifts and fluorescence quenching in different acetone (organic solvent) and water mixtures reflect a general effect on the hydrogen-bonding environment of the fitc through water-acetone hydrogen bridges and specific effects due to direct fitc -acetone hydrogen bonding. by increasing acetone concentrations, the fitcwater connections change to fitc-acetone interactions, resulting in basic modifications of hydrogen bonds due to fluorescence quenching. both the absorption spectra and fluorescence properties (quenching) appear to be dominated by increasing hydrophobicity, indicating that the spectral shifts of the fitc can be used as an indicator of its hydrogen-bonding environment. application of fluorescein as a probe of hydrogen bonding in the microenvironment immediately surrounding the fluorophore illustrated the effect with reference to the fluorescein-anti-fluorescein antibody complex (30). this finding fits well in our results (as demonstrated in figure 2, figure 6, and figure 7), where it appears that binding sites of our anti-fitc monoclonal antibody and the recognized antigen (fitc) are increasingly dehydrated and become hydrophobic during the immunocomplex formation. hydrophobic interactions between the antigen and antibody molecules seem to be essential in the physico-chemical event of antigen-antibody reaction, which is supported both by our fluorescent spectroscopic and fluorescent polarization measurements and other data from the relevant literature (15, 23, 30, 31). our model experiment, involving non-polar solvent and water mixtures, supported our hypothesis about the importance of hydrophobic interactions in the stabilization of the antigen-antibody ligands. however, further experimental evidence is needed. the results of the infrared spectroscopic analysis can prove the hydrophobic nature of immunobinding between anti-fitc and fitc. this hydrophobic interaction also perturbed the electron cloud of the polyaromatic cyclical structure. these results can also prove the quenching effect caused by anti-fitc monoclonal antibody. our fluorescent and infrared spectroscopic data suggest that the hydrophobic connections not only participate in the stabilization of antigen-antibody complexes, but also modify the physico-chemical nature of participating molecules. the main ligand-induced conformational changes on the immunoglobulin molecules causing a basic modification of their functions are well-known. however, the same physicochemical influences on the fine structure of antigens have been less analyzed so far due to technical difficulties (9, 10, 11). in the original fitc – anti-fitc model, the fluorescence-quenching effect was observed during antigen-antibody reactions (9, 32). the fluorescent activity of fitc molecules as antigens in this model system seems to be beneficial for studying fine structural changes in the antigen after binding with a specific immunoglobulin (9, 33). using fluorochrome-labelled anti-idiotypic antibodies and fluorescent spectroscopic measurements, experimental data published suggest the importance of water molecules in the ligand formation (2). our data, together with previous findings, help understand the molecular dynamics of immunobinding (2, 3, 7). the immunocomplex formed by hydrophobic regions of a specific epitope on the antigen and the recognition part on the antibody molecule stabilized with the surrounding hydrate shell seems to be thermodynamically strong enough for stable molecular binding without a covalent chemical interaction (34, 35). in our model, direct observations were possible for the physico-chemical changes on the bound antigen because the quenching effect of fitc fluorescence during the immunocomplex formation was analyzed by different and relevant spectroscopic techniques. our pre-sent fluorescent polarization and infrared spectroscopic measurements reflect a local decrease in the hydration degree in the seemedj 2022, vol 6, no. 2 physico-chemical background of antigen-antibody reactions 17 southeastern european medical journal, 2022; 6(2) submolecular area of a specific ligand between the small antigen (hapten) molecule and the hypervariable region of igg. this “exclusion” of water molecules from the binding surfaces occurs due to a dramatic modification of the molecular microenvironment. changes in hydration can influence the functions of the immunoglobulins and the physico-chemical appearance of the antigen at the same time (36). participation of antigens in immunocomplex formation can cause altered immunoreactivity in further physiological and pathological immune reactions. acknowledgement. this paper is a product of our long scientific discussions with professor béla somogyi†. i am grateful for his suggestions and technical help. many thanks to gergely nagy for the initial fluorimetric measurements, to professor miklós nyitrai for the analysis and discussion of the results of fluorescence polarization measurements, and to dr. tamás lóránd for the infrared spectroscopy measurements and their evaluation. disclosure funding. this paper was realized with the financial support of the university of pécs, medical school (pécs, hungary), and the following grants: the national science foundation of hungary (otka t020661), national committee for technical development of hungary (omfb 95-97-48-1028), and by health science committee of hungary (ett -06 401/96). transparency declaration: we declare that we have no commercial or potential competing interests or any financial and personal relationships with other people or organizations that could inappropriately influence our network. conflict of interest: none declared. references 1. davies dr, padlan ea, sheriff s. antibodyantigen complexes. annu rev biochem. 1990;59:439-73. doi: 10.1146/annurev.bi.59.070190.002255. 2. weidner km, denzin lk, voss ew jr. molecular stabilization effects of interactions between anti-metatype antibodies and liganded antibody. j biol chem. 1992 may 25;267(15):10281-8. 3. wilson ia, stanfield rl. antibody-antigen interactions: new structures and new conformational changes. curr opin struct biol. 1994 dec;4(6):857-67. doi: 10.1016/0959440x(94)90267-4. 4. edmundson ab, guddat lw, shan l, fan zc, hanson bl. structural aspects of conformational changes in ligand binding by antibody fragments. res immunol. 1994 jan;145(1):56-61. doi: 10.1016/s09232494(94)80045-6. 5. dyson hj, wright pe. antigenic peptides. faseb j. 1995 jan;9(1):37-42. doi: 10.1096/fasebj.9.1.7821757. 6. haaijman jj, coolen j, kröse cj, pronk gj, ming zf. fluorescein and tetramethyl rhodamine as haptens in enzyme immunohistochemistry. histochemistry. 1986;84(4-6):363-70. doi: 10.1007/bf00482964. 7. bhat tn, bentley ga, boulot g, greene mi, tello d, dall'acqua w, souchon h, schwarz fp, mariuzza ra, poljak rj. bound water molecules and conformational stabilization help mediate an antigen-antibody association. proc natl acad sci u s a. 1994 feb 1;91(3):1089-93. doi: 10.1073/pnas.91.3.1089. 8. laver wg, air gm, webster rg, smithgill sj. epitopes on protein antigens: misconceptions and realities. cell. 1990 may 18;61(4):553-6. doi: 10.1016/00928674(90)90464-p. erratum in: cell 1990 aug 10;62(3):following 608 9. mummert me, voss ew jr. effects of secondary forces on the ligand binding properties and variable domain conformations of a monoclonal anti-fluorescyl antibody. mol immunol. 1996 sep;33(13):1067-77. doi: 10.1016/s0161-5890(96)00066-1. seemedj 2022, vol 6, no. 2 physico-chemical background of antigen-antibody reactions 18 southeastern european medical journal, 2022; 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6(2) 29. mummert me, voss ew jr. secondary force-mediated perturbations of antifluorescein monoclonal antibodies 4-4-20 and 9-40 as determined by circular dichroism. j protein chem. 1998 apr;17(3):237-44. doi: 10.1023/a:1022532618038. 30. müller jd, nienhaus gu, tetin sy, voss ew. ligand binding to anti-fluorescyl antibodies: stability of the antigen binding site. biochemistry. 1994 may 24;33(20):6221-7. doi: 10.1021/bi00186a023. 31. klonis n, clayton ah, voss ew jr, sawyer wh. spectral properties of fluorescein in solvent-water mixtures: applications as a probe of hydrogen bonding environments in biological systems. photochem photobiol. 1998 may;67(5):500-10. 32. watt rm, voss ew jr. mechanism of quenching of fluorescein by anti-fluorescein igg antibodies. 1977. immunochemistry. jul;14(7):533-51. doi: 10.1016/00192791(77)90308-1. pmid: 303233 33. omelyanenko vg, jiskoot w, herron jn. role of electrostatic interactions in the binding of fluorescein by anti-fluorescein antibody 4-420. biochemistry. 1993 oct 5;32(39):10423-9. doi: 10.1021/bi00090a018. 34. howlett jr, ismail aa, armstrong dw, wong pt. pressure-induced conformational changes in an antigen and an antibody and the implications on their use for hyperbaric immunoadsorption. biochim biophys acta. 1992 oct 20;1159(3):227-36. doi: 10.1016/01674838(92)90049-j. 35. sii d, sadana a. bioseparation using affinity techniques. j biotechnol. 1991 jun;19(1):83-98. doi: 10.1016/01681656(91)90076-8. 1 author contribution. single author seemedj 2023, vol 7, no 1 automated elisa method for hep-25 36 southeastern european medical journal, 2023; 7(1) original article verification of the automated elisa assay for hepcidin-25 in human serum 1 tara rolić 1,2*, sanja mandić 1,2, iva lukić 1,2, vesna horvat 1,3, ines banjari 4 1 department of chemistry, biochemistry and clinical chemistry, faculty of medicine, university of osijek, osijek, croatia 2 institute of clinical laboratory diagnostics, osijek university hospital, osijek, croatia 3 medical biochemistry laboratory, health center osijek-baranja county, osijek, croatia 4 department of food and nutrition research, faculty of food technology osijek, osijek, croatia *corresponding author: tara rolić, tara.rolic@kbco.hr received: feb 20, 2023; revised version accepted: mar 20, 2023; published: apr 30, 2023 keywords: enzyme-linked immunosorbent assay, hepcidins, homeostasis, iron abstract introduction: hepcidin-25, the bioactive form of hepcidin, is the master protein in regulating iron homeostasis. serum concentrations, measured by different methods, are often incomparable and complicate results interpretation. materials and methods: the aim was to verify the first fully automated enzyme-linked immunosorbent assay (elisa) method, using the drg hybrid xl analyzer (drg instruments, marburg, germany) standardized against the mass spectrometry method. intra(cvi) and inter-assay (cvg) precision and bias were performed using commercially available controls with low (c1) and high (c2) concentrations. the reference interval was verified by analyzing serum samples of 20 healthy males. results: cvi = 9.1% (c1), 4.5% (c2); cvg = 8.9% (c1), 5.6% (c2); calculated bias was 33% for c1 and 20% for c2, respectively. conclusion: verification of the fully automated elisa method for hepcidin-25 in serum on the drg hybrid xl analyzer met the analytical acceptance criteria. (rolić t, mandić s, lukić i, horvat v, banjari i. verification of the automated elisa assay for hepcidin25 in human serum. seemedj 2023; 7(1); 36-41) seemedj 2023, vol 7, no 1 automated elisa method for hep-25 37 southeastern european medical journal, 2023; 7(1) introduction hepcidin-25, the bioactive 25 amino acid peptide is the master iron-regulatory hormone and is predominantly generated in the liver by proteolytic cleavage of prohepcidin at the cterminus (1). n-terminal removal of hepcidin-25 can result in short peptides of 20-24 amino acids which have reduced activity but can interfere with some immunoassays (2). hepcidin-25 is the only active form of hepcidin in serum and has the main role in iron status regulation by downregulating the expression of ferroportin. hepcidin binds to the cell surface iron transporter – ferroportin – inducing its internalization and degradation (3). the result is increased intracellular iron storage, decreased dietary iron absorption and decreased serum iron concentration (1). in conditions of increased demand for iron, (iron deficiency, hypoxia, anemia and erythropoiesis) hepatocellular hepcidin synthesis decreases (4, 5). hepcidin deficiency causes hereditary hemochromatosis, characterized by body iron overload that may progress into liver cirrhosis (6). in addition, low hepcidin-25 concentration can be induced in iron-loading anemias and chronic hepatitis c (7, 8). in contrast, hepcidin synthesis is induced by inflammation and infection (9). high hepcidin-25 concentration has been found in iron-refractory iron-deficiency anemia, during infection, chronic kidney disease as well as in cancer (9–11). therefore, hepcidin-25 serum levels are valuable for identifying and differentiating specific diseases or conditions related to iron homeostasis (7, 10, 12). so far, two main techniques have been used for serum hepcidin determination: mass spectrometry (ms) and immunochemistry – solid phase enzyme-linked immunosorbent assay (elisa) (13–15). immunochemistry assays are commonly used to carry out routine tests in laboratories. however, the elisa method is not suitable for individual and random-access testing because results provided by different methodologies show high discrepancies. analytical method verification is required for providing objective evidence that an analytical procedure meets the requirements suitable for scientific research and routine application in the laboratory (16). in 2016, the first fully automated immunoassay, an important step forward, was introduced to the market. until then, only manual immunoassays were available. the aim of this study was to verify in a clinical laboratory the fully automated elisa method validated by drg company by ms (results not published) for measuring concentrations of hepcidin-25 in human serum. materials and methods subjects and blood sampling commercially available hepcidin samples were obtained from drg company (drg instruments, llc (gmbh), marburg, germany): c1-normal level (4.6 µg/l) and c2-high level (44.3 µg/l), were used as standards for the verification procedure. additionally, blood samples from healthy subjects (regular male blood donors aged 18 years or older), were collected in august 2020 at the department of transfusion medicine, osijek university hospital, following the guidelines on venipuncture for blood donation. all subjects were previously screened for anemia at the same department according to the guidelines for blood donors. the exclusion criteria were hemoglobin concentration of less than 135 g/l. inclusion criteria were iron, ferritin and transferrin levels within the reference range. indicators of iron status were conducted at the institute of clinical laboratory diagnostics. informed consent was signed by all participants. venipuncture was performed, and blood was collected using tubes (5 ml) (becton, dickinson and company, franklin lakes, nj, usa) without anticoagulants. the tubes were centrifuged for 10 minutes at 2,000 g and the serum was used for measuring the concentration of hepcidin-25. seemedj 2023, vol 7, no 1 automated elisa method for hep-25 38 southeastern european medical journal, 2023; 7(1) analytical methods analytical verification of the method was performed according to the clinical and laboratory standards institute (clsi) guideline ep15-a2 on method verification (16). method verification procedure included the assessment of the accuracy of series (repeatability) and dayto-day (intermediate precision) accuracy, measurement uncertainty and reference interval verification. after reconstituting the lyophilized control samples following the manufacturer’s instructions, verification steps were performed. control standard samples were analyzed in triplicate for five consecutive days and the data were used to calculate both accuracy and variability. the normal reference range was checked from 20 serum samples from healthy blood donors. hepcidin-25 concentrations were measured by the automated elisa method using the drg hybrid xl (drg instruments, llc (gmbh), marburg, germany) analyzer. the manufacturer declared that the hepcidin-25 elisa method was standardized against the mass spectrometry method. all data were calculated using the excel program: mean value, standard deviation, coefficient of variation and bias according to the formulas provided in supplement table 1. reference interval verification was performed using medcalc for windows, version 12.4.0.0 (medcalc software, mariakerke, belgium). the reference interval provided by the manufacturer was confirmed in serum samples. the distribution of the results was normal, calculated data obtained in excel were used for the reference interval verification – the interval in which the central 95% values of a healthy subject lie. the used limits of normality: mean ± 1.96 x sd. results in this research, we performed method verification in terms of intraand inter-assay precision and bias assessment. method verification was performed by analyzing two concentration levels of commercial controls. results are summarized in table 1. we confirmed the declared inter-assay precision for both concentration levels considering the concentration values. namely, < 10.7% and < 10.0% for normal (c1) and high (c2) control levels, respectively. calculated bias was higher in c1 (> 30%) than in c2 (20%). table 1. coefficients of variation for control samples cvi cvg total cv bias c1 9.0% 8.9% 11.6% 33% c2 4.5% 5.6% 6.7% 20% c1=control sample level 1; c2=control sample level 2; cvi=intraindividual coefficient of variation; cvg=interindividual coefficient of variation; cv=coefficient of variation the mean hepcidin-25 level in serum samples was 10.2 µg/l (sd 9.9) which is within the normal reference range stated by the manufacturer (drg instruments, llc (gmbh), marburg, germany) declared for the 2.5th and 97.5th percentile as of 0.2–34.4 µg/l obtained on 20 healthy male blood donors in the current study. seemedj 2023, vol 7, no 1 automated elisa method for hep-25 39 southeastern european medical journal, 2023; 7(1) supplemental table 1: measured values of control samples during five consecutive days in triplicate 1. intraand interassay precision control sample level 1 day: 1 2 3 4 5 1 5,25 5,95 5,87 5,70 6,92 2 6,25 5,90 6,13 5,93 7,08 3 5,38 5,20 7,54 5,47 6,29 mean 5,63 5,68 6,51 5,70 6,76 sd 0,54 0,42 0,90 0,23 0,42 sr 0,55 cv% 9,06 grand mean 6,06 xd grand mean 0,19 0,14 0,21 0,13 0,50 sb 0,54 cv% 8,90 sl 0,70 cv% 11,56 2. intraand interassay precision control sample level 2 1 2 3 4 5 1 54,30 49,70 59,70 54,10 53,70 2 55,30 56,00 60,80 56,20 53,50 3 52,30 51,00 59,10 61,90 54,80 mean 53,97 52,23 59,87 57,40 54,00 sd 1,53 3,33 0,86 4,04 0,70 sr 2,49 cv% 4,48 grand mean 55,49 xd grand mean 2,33 10,63 19,13 3,64 2,23 sb 3,08 cv% 5,55 sl 3,69 cv% 6,65 sd= standard deviation; cv= coefficient of variation; sb= standard grand mean deviation; sr= mean standard deviation; si= total standard deviation discussion a study designed by aune et al. described a framework for optimizing hepcidin measurement and improvements in method standardization (17). prior to the aune et al. study, diepeveen et al. standardized hepcidin methods (18). both studies proposed an international standard for methods calibration. aune et al. compared 16 different methods for hepcidin and obtained a mean accuracy of 145% (ci 76–540%), for all methods (17). interestingly, among 9 ms and 7 elisa methods, and described here, the elisa drg hybrid xl bioactive hepcidin-25 method, the declared accuracy was 125% (ci 109–147%). other elisa methods in the aune et al. study had similar accuracies with only one method having a higher accuracy (340% (ci 274– 540%)). the lack of standardization is reflected by the large variation in results. it is important to note that only hepcidin-25 is the biologically active isoform to be measured, but with elisa methods there is a certain rate of cross-reactivity seemedj 2023, vol 7, no 1 automated elisa method for hep-25 40 southeastern european medical journal, 2023; 7(1) with other hepcidin isoforms, usually declared by the manufacturer. in the current study, the optimal minimum cv for precision was 2.27% and 1.12%, calculated as cvi x 0.25 pointing out that the elisa drg hybrid xl method has optimal cv when compared to the literature data (< 12.2%) (19). we must point out that the available data about hepcidin-25 biological variation were obtained on mice and a few human samples, in a study by murphy et al. (19). the obtained intraindividual biological variation of hepcidin-25 was 48.8% while the inter-individual coefficient of variation was 154.1%. here described study obtained cvi and cvg around 10%. the main advantages of the automated elisa method are straightforward procedure, high sensitivity, ready-to-use reagents, short analysis time (2 hours), small sample volume (160 µl), and the possibility of individual sample analysis compared to ms analysis which is multiplex and delays in results. a significant advantage of this method is its standardization against the ms. there are limitations to this current study. the drg company reagent manufacturer did not report the desirable bias or inaccuracy for the method. also, the drg company used patient’ samples for the initial method validation, while our study verification was performed with commercial control samples so there is a difference in the matrix. in addition, we did not compare the drg method with other hepcidin25 methods. for the verification, we used control samples provided by the manufacturer. therefore, we could not compare our data with the manufacturer’s validation data which were conducted on the patient samples. during the verification study, we were not able to perform commercial controls of a third-party manufacturer. finally, the reference interval verification was performed using only serum samples of healthy males. conclusion the verification of the fully automated elisa method for determining hepcidin-25 levels in serum samples performed on the drg hybrid xl analyzer showed desirable analytical reproducibility and met all acceptance criteria. acknowledgement. hepcidin reagents and supplies were provided free of charge by the limited liability company (gmbh) drg diagnostics. all authors do not have any conflicts of interest. the authors would like to thank the local distributor (croatian) of drg diagnostics, jasika company and mr. hrvoje štimac, for his generous donation, support and contribution, as well as prof. dr. sc. george y wu for detailed revision of the paper. disclosure funding. no specific funding was received for this study. competing interests. none to declare. list of abbreviations: ms = mass spectrometry elisa = solid phase enzyme-linked immunosorbent assay c1 = control sample level 1 (normal value) c2 = control sample level 2 (high value) clsi = clinical and laboratory standards institute cvi = intraindividual coefficient of variation cvg = interindividual coefficient of variation cv = coefficient of variation sd = standard deviation ci = confidence intervals references 1. ganz t., the discovery of the iron-regulatory hormone hepcidin. clin chem 2019; 65:1330-31; doi: 10.1373/clinchem.2019.306407 2. abbas im, vranic m, hoffmann h, el-khatib ah, montes-bayón m, möller hm. et al. investigations of the copper peptide hepcidin-25 by lc-ms/ms and nmr. int j mol sci 2018; 19:2271; doi: 10.3390/ijms19082271 3. billesbølle cb, azumaya cm, kretsch rc, power as, gonen s, schneider s et al. structure of hepcidinseemedj 2023, vol 7, no 1 automated elisa method for hep-25 41 southeastern european medical journal, 2023; 7(1) bound ferroportin reveals iron homeostatic mechanisms. nature 2020; 586:807–11; doi: 10.1038/s41586-020-2668-z 4. muckenthaler mu, rivella s, hentze mw, galy b. a red carpet for iron metabolism. cell 2017; 168:344-61; doi: 10.1016/j.cell.2016.12.034 5. papanikollaou g, pantopoulus k. systematic iron homeostasis and erythropoiesis. iubmb life 2017; 69:399-413; doi: 10.1002/iub.1629 6. pantopoulos k., inherited disorders of iron overload. front nut, 2018; 5; doi: 10.3389/fnut.2018.00103 7. d’angelo g. role of hepcidin in the pathophysiology and diagnosis of anemia. blood res 2013; 48:10-5; doi: 10.5045/br.2013.48.1.10 8. ali m, sayed a, elhawary m, assem a, serum hepcidin in patients with chronic hepatitis c and its relation to treatment with antiviral drugs. aimj, 2020; 1:152-57; doi: 10.21608/aimj.2020.28247.1201 9. weiss g, ganz t, goodnough lt. anemia of inflammation. blood, 2019;133:40-50; doi: 10.1182/blood-2018-06-856500 10. aksan a, farrag k, aksan s, schroeder o, stein j. flipside of the coin: iron deficiency and colorectal cancer. front immunol 2021; 12:635899; doi: 10.3389/fimmu.2021.635899 11. santos-silva a, ribeiro s, reis f, belo l, hepcidin in chronic kidney disease anemia. vitam horm 2019; 110:243-64; doi: 10.1016/bs.vh.2019.01.012 12. yiannikourides a, latunde-dada go. a short review of iron metabolism and pathophysiology of iron disorders. medicines (basel) 2019; 6:85; doi: 10.3390/medicines6030085 13. enko d, zelzer s, fauler g, herrmann m, evaluation of a commercial liquid-chromatography highresolution mass-spectrometry method for the determination of hepcidin-25. biochem med (zagreb) 2019; 29:020701; doi: 10.11613/bm.2019.020701 14. abbas im, hoffmann h, montes-bayón m, weller mg, improved lc-ms/ms method for the quantification of hepcidin-25. anal bioanal chem 2018; 410:3835-46; doi: 10.1007/s00216-018-1056-0 15. uelker b, cushman i, dudek t, herkert m, geacintov ce, high sensitivity hepcidin-25 bioactive elisas: manual and fully automated system for the quantification of hepcidin-25 in human serum and plasma (abstract). blood 2016; 128:4828 16. clsi. user verification of precision and estimation of bias: approved guideline third edition. wayne, pa: clsi; 2014. clsi document ep15a3 17. aune et, diepeveen le, laarakkers cm, klaver s, armitage ae, bansal s, et al., optimizing hepcidin measurement with a proficiency test framework and standardization improvement. clin chem lab med 2020, 59:315-323; doi: 10.1515/cclm-2020-0928 18. diepeveen le, laarakkers cmm, martos g, pawlak me, uguz ff, varberne kesa et al. provisional standardization of hepcidin assays: creating a traceability with a primary reference material, candidate reference method and commutable secondary reference material. clin chem lab med 2018; 57:864:872; doi: 10.1515/cclm-2018-0783 19. murphy at, witcher dr, luan p, wroblewski vj. quantitation of hepcidin from human and mouse serum using liquid chromatography tandem mass spectrometry. blood 2007; 110:1048-54; doi: 10.1182/blood-2006-11-057471 author contribution. acquisition of data: tr administrative, technical or logistic support: tr analysis and interpretation of data: tr conception and design: tr critical revision of the article for important intellectual content: tr drafting of the article: tr final approval of the article: tr guarantor of the study: tr obtaining funding: tr provision of study materials or patients: tr seehsj south eastern europe health sciences journal southeastern european medical journal seemedj faculty of medicine osijek josip juraj strossmayer university of osijek j. huttlera 4 croatia – 31000 osijek seemedj@mefos.unios.hr seemedj.mefos.unios.hr editorial seemedj 2022;6(2): 1-85 dear colleagues, hereby, new 12th issue of seemedj is presented to you. this issue brings 7 articles with various topics. the molecular dynamics of the antigen-antibody reaction and the role of water molecules is presented in the article by nemeth p. iskandarov et al investigated bioactive components that affect blood coagulation system by fractionation of vipera berus berus snake venom which may be utilized in biotechnology and medicine. microcirculatory blood flow after acute exertional exercise was examined by potočnik n. tff3 is involved in the pathogenesis of sepsis, but the exact mechanism is not yet clear. this issue has been reviewed by bazina and baus lončar. comorbidities of psychiatric and somatic disease pose a major challenge to modern medicine and the multidisciplinary approach and cooperation in treatment are necessary to improve the patients’ quality of life and life expectancy, as presented in a work by marušić r et al. vidović et al detected vitamin d deficiency in a population of medical students, which is significant problem noted elsewhere also. and finally, krajina kmoniček et al reviewed the effects of iron chelators in covid-19 disease. we are happy to inform our potential authors that seemedj has been included to another citation bases: crossref, dimensions and publons, thus widening our accessibility to readers. the art work at the cover page is authored by nevenka arbanas, croatian graphic artist born in batina, baranja. this way, we continue to present our national but widely recognized artists with the origin from easter slavonia and baranja. i hope that readers will find relevant published articles for their work. on the behalf of the editorial board and my own, i warmly greet our readers and invite them to join us in the endeavor of publishing their own scientific work in seemedj. ines drenjančević, md, phd editor-in-chief southeastern european medical journal (seemedj) seemedj 2017, vol 1, no. 1 intrinsic control and environmental factors in food consumption related to obesity 121 southeastern european medical journal, vol 1, 2017. intrinsic control and environmental factors in food consumption related to obesity 1 ivana jukić1, aleksandar kibel1,2, dijana kibel1,3, ines drenjančević1 1 department of physiology and immunology, faculty of medicine, josip juraj strossmayer university of osijek, osijek, croatia 2 department for cardiovascular diseases, osijek university hospital, osijek, croatia 3 department of diagnostic and interventional radiology, osijek university hospital, osijek, croatia corresponding author: ivana jukić, md, phd igrizelj@mefos.hr epidemiology of obesity increased accumulation of fat tissue leads to obesity. the most common ways to evaluate received: april 21, 2017; revised version accepted: may 10, 2017; published: may 25. 2017 keywords: obesity, pomc, melanocortins, chronic stress, social behavior obesity are by the calculation of the body mass index (bmi), the measurement of waist circumference (wc) and the calculation of the ratio of the circumference of waist and hip abstract obesity results from a complex interaction of genetic, hormonal, physiological, anatomical, psychological, behavioral and environmental factors causing an imbalance between energy intake and energy expenditure. according to the world health organization, the estimated number of obese people around the world has doubled from 1980, meaning that more than 600 million people worldwide are obese. obesity is associated with low-level chronic inflammation and represents a major risk factor for cardiovascular and metabolic diseases, but also some cancers. centers that regulate food intake and energy balance are placed in the hypothalamus. chemical signals are transmitted between hypothalamic neurons, and those neurons also affect the secretion of different hormones that are important for maintaining energy balance and metabolism. moreover, genetic predisposition is also a risk factor for obesity development. key neuronal populations for maintaining energy balance are the orexigenic agouti related peptide (agrp)/neuropeptide y (npy) neurons and the anorexigenic proopiomelanocortin (pomc) neurons. this review attempts to present the prevalence and the major pathways regulating energy balance that may be affected by many environmental and social factors, such as emotions and human behavior, and can lead to obesity. (jukić i, kibel a, kibel d, drenjančević, i. intrinsic control and environmental factors in food consumption related to obesity. seemedj 2017;1(1);121-129) seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 122 southeastern european medical journal, vol 1, 2017. (whr). a bmi of 20-25 kg/m2 is considered normal; 25-30 kg/m2 is overweight; 30-40 kg/m2 is defined as obesity and >40 kg/m2 is defined as extreme obesity. for children and adolescents, a bmi >95 percentile is considered overweight/obesity (1). a waist circumference (wc) of at least 88 cm for women and 102 cm for men is defined as obesity (2). a whr above 1.0 in male subjects and above 0.6 in women is considered as an obesity pattern (3). the prevalence of obesity in the general population is increasing worldwide. based on the nutrition examination survey (nhanes) 1999–2000 in the usa, the prevalence of overweight in adults was 64.5% and the prevalence of obesity was 30.5% (4). the most comprehensive overview of the prevalence of obesity is provided by the world health organization (who) global status report on noncommunicable diseases 2014 (5), which showed that the age-standardized prevalence of obesity in adults aged 18 years and over (cutoff point set to bmi ≥30 kg/m2) in the usa was 33.7%. canada had a prevalence of obesity of 28%, mexico 28.1%, argentina 26.3% and brazil 20%. the same publication reported that in europe, the prevalence of obesity among the general population is as follows: uk 28.1%, slovenia 25.1%, spain 23.7%, croatia 23.3%, italy 21%, germany 20.1% turkey 29.5, france 23.9% and bosnia and herzegovina 17.9%. interestingly, in asia the prevalence of obesity was significantly lower: the prevalence of obesity was in china 6.9%, india 4.9% and japan 3.3%. australia has a prevalence of obesity in the general population of 28.6%. in africa, there is a wide range of obesity prevalence, from 28.9% in egypt to 10.5% in zimbabwe, 11% in congo and 11.4% in cameroon (5). based on who data, in 2014 more than 1.9 billion adults aged 18 years and older (39% [38% of men and 40% of women]) were overweight. of these over 600 million adults were obese—about 13% of the world’s adult population (11% of men and 15% of women). the worldwide prevalence of obesity more than doubled between 1980 and 2014 (5). the prevalence of obesity is linked to the prevalence of various cardiometabolic diseases, such as diabetes mellitus. recently, in 44 observational or clinical studies that were evaluated in a systematic literature review by colosia et al, the obesity prevalence in t2dm subjects was determined based on bmi or wc (2). based on bmi, the prevalence of obesity in that population group was 30-85.5% in asia, 32.764.1% in europe, 26.8-64.3% in north america, 32.5-59% in south america and 32-42.5% in africa. the prevalence of obesity in t2dm patients based on waist circumference was 56-67% in europe, 54.6% in africa and 20-81% in asia. however, there were no studies taking into account obesity based on wc in that publication, and not all countries were covered by the examined studies (2). due to its effects on hormonal balance, autonomic system function and various organ functions, obesity represents a significant risk factor for many other chronic diseases such as hypertension, atherosclerosis, diabetes mellitus and chronic kidney disease.. the pathophysiological mechanism of obesity the development of obesity is a consequence of several pathophysiological factors. first of all, it is an imbalance of food intake and energy expenditure. the central control of energy balance by the hypothalamus and the feedback loop of peripheral metabolic factors from the gastrointestinal and endocrine system, including adipose tissue, are very important factors, particularly in obesity related to chronic stress and other cardiometabolic diseases. genetic predisposition accounts for approximately 30% of the risk for obesity development (6). the most recognized mutations are those of receptors or hormones related to the regulation of feeding behavior, and subsequently energy balance: e.g. mutations of the leptin gene and the leptin receptor gene, the mutation of the proopiomelanocortin gene (pomc) and mutations of genes for melanocortin receptors (e.g. mcr-4) (7). these conditions lead to pathological obesity with other metabolic and hormonal imbalances. central control of dietary intake (energy balance) an individual’s energy intake and expenditure must be balanced over time to ensure adequate seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 123 southeastern european medical journal, vol 1, 2017. body composition and function (8). the quantity of food intake is regulated on a short-term basis, which controls the intake of food at each meal, and long-term, which is mainly concerned with the maintenance of normal quantities of body energy stores (9). the main homeostatic system involved in the regulation of energy balance is located in the hypothalamus, and the nucleus arcuatus has a central role in these processes. it functions by integrating neural and nutrient signals with hormonal signals that arise in the small intestine, liver, pancreas, adipose tissue and brainstem (10). the total daily energy intake is a function of the size, frequency, and qualitative composition of meals, and the perception of hunger and the decision to initiate a meal are determined by complex interactions between genetic, social, learned, environmental, circadian, and humoral factors (11). centers for the control of hunger and satiety are located in the hypothalamus, stimulation of the lateral nuclei causes hyperphagia (their destruction has the opposite effect), while stimulation of the ventromedial nuclei causes satiety and aphagia (8). other areas add to the complexity: paraventricular nuclei destruction leads to excessive eating, dorsomedial nuclei destruction leads to depressed eating, and arcuate nuclei are centers where several hormonal signals from the adipose tissue and gastrointestinal tract are integrated to regulate food intake (9). in addition to these main structures of the homeostatic system, an important role is played by the brain’s reward system — located in proximity to the hypothalamus with its main nodes centered on the ventral striatum. it includes the nucleus accumbens, the ventral pallidum, and the ventral tegmental area (10). evolutionary development led to the emergence of protective central mechanisms that are focused on the resistance of fat loss and on the maintenance of body weight, crucial features that helped enable the survival of the human species throughout time (12). unfortunately, such a system predisposes humans to obesity in times of abundance, with all the known adverse effects. obesity has become one of the leading medical challenges of the 21st century (11). “hunger hormones,” like orexin and ghrelin, as well as high-calorie food, incite individuals to eat, while “satiety hormones” like leptin, insulin and other so called “brain-gut peptides,” suppress feeding behavior (12). weight increase and obesity can result from long-term imbalance between the hunger and satiety signals (12). there exist two neuronal populations that are crucial in energy balance homeostasis and are located in the arcuate nucleus of the hypothalamus: the orexigenic agouti related peptide (agrp)/neuropeptide y (npy) neurons and the anorexigenic proopimelanocortin (pomc) neurons (10). activation of the pomc neurons decreases food intake and increases energy expenditure. on the other hand, activation of the npy-agrp neurons exerts opposite effects, increasing food intake and reducing energy expenditure, with both of these neuronal groups having significant interaction (8). these neuronal groups are the main targets of hormones that regulate appetite, including leptin, ghrelin, insulin, cholecystokinin and others. pomc neurons secrete the alphamelanocyte-stimulating hormone (msh), which acts via several types of melanocortine receptors (mcr) and through them leads to a reduction in food intake and an increase in energy expenditure (9), as discussed below. agrp are antagonists of mcr, increasing food intake. npy increases appetite and is produced when energy reserves in the body are low (9). chronic stress and obesity stress is an incentive that occurs in response to certain experiences, representing the state of disrupted hemostasis. the organism reacts to stress via the central and peripheral segments of the autonomic nervous system and via the hypothalamic-pituitary-adrenal (hpa) pathway. response to stress can be acute condition, and is necessary for the maintenance of homeostasis, or chronic stress, which is prolonged and may cause some disease states. acutely, within a few hours, glucocorticoids act to inhibit the secretion of crh and acth. that physiological effect is noticeably altered in seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 124 southeastern european medical journal, vol 1, 2017. chronic stress, where the effect of glucocorticoids on the brain is excitatory (13-15). many recent studies have shown that chronic stress may contribute to an increased risk for the development of obesity and other metabolic diseases. one of the elementary responses to stress is mediated by the activation of the paraventricular nucleus of the hypothalamus that secretes a corticotrophin-releasing hormone (crh), which stimulates the secretion of adrenocorticotrophin (acth) from the anterior pituitary gland. acth binds to its mc2r receptors on the adrenal cortex and thus stimulates the secretion of cortisol (13-15). cortisol causes the elevation of blood glucose and insulin concentration, whose long-term effect may cause insulin resistance and diabetes mellitus type 2. the long-term effect of chronic stress on the hpa axis leads to increased accumulation of visceral fat, but it also has been shown that obesity represents a state of chronic systemic low-grade inflammation that may cause an impaired function of the hpa axis. in the state of chronic stress, glucose metabolism is disrupted. the increased secretion of acth and hence a high level of glucocorticoids, primarily cortisol, is associated with weight gain and the high production of proinflammatory hormones and adipokines by adipose tissue depots (15-17). the main central targets of insulin (and leptin) action are the pomc and agrp neurons of the arcuate nucleus of the hypothalamus. some studies suggest a major physiological importance of insulin receptors in the brain in the long-term modulation of energy balance, whereas the disturbance of the insulin receptor figure 1. chronic stress and central mechanisms of weight gain due to constant activation of the hpa axis during chronic stress, there is a marked increase in secretion of pomc, acth and glucocorticoids, which leads to enhanced blood glucose levels and increased insulin secretion. persistent elevated insulin levels decreases sensitivity or number of insulin receptor in the npy/agrp region and reduce inhibition of the orexigenic neurons that leads to obesity. seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 125 southeastern european medical journal, vol 1, 2017. gene may cause increased body fat and high plasma levels of insulin and leptin (18). chronic stress also increases the activation of the sympathetic nervous system, and thus contributes to impaired glucose tolerance and to an increased risk for developing of cardiovascular events (19). thus, chronic stress that activates the hpa axis, as well as the sympathetic nervous system, may progressively contribute to the occurrence of obesity and metabolic syndrome (figure 1). the role of msh receptors it is well accepted that the central melanocortin signaling pathway has a critical role in the maintenance of energy balance, and it is known that peptides generated from pomc have a key role in controlling food intake and weight gain (20, 21). melanocortin receptors present a distinct family of g-protein-coupled receptors; they are coupled to adenylate cyclase and exert their effects primarily by activating a campdependent signaling pathway (20). melanocortin receptors are differently distributed in various kinds of tissue and differ from each other in their affinity for binding various melanocortins and/or their antagonists, agouti signaling protein (asp) and agouti-related protein (agrp) (20). prohormone convertase 1 (pc1) is expressed in pituitary corticotrophs and produces acth, while the expression of pc1 and prohormone convertase 2 (pc2) within the hypothalamus leads to the production of α-, βand γ-msh (7). the actions of acth and melanocortins are mediated by specific interactions with five melanocortin receptors (mc1r to mc5r), two of which, mc3r and mc4r, are predominantly expressed within the central nervous system, and in the context of human energy balance and body weight regulation, they— especially mc4r— have the most important role (7,22). mc1r is expressed in pigment-producing cells of the skin and hair, and when α-msh binds to it the production of pigment melanin is stimulated. also mc1r is expressed on cells of the immune system, wherefore α-msh has antiinflammatory effects (7). mc2r is an acth receptor expressed in the adrenal cortex, while mc3r is expressed in the brain (mainly in the hypothalamus, cortex, thalamus and hippocampus), and over it γ-msh plays a role in cardiovascular functions and sodium and energy homeostasis (7). the β-msh peptide performs its action in weight regulation by binding to mc4r, helping to maintain energy balance (7), and in this context has the most important role. mc5rs are expressed at low levels in numerous tissues, including the sebaceous gland, where it has a role in sebaceous secretion (7). it is thought that the anorectic effect of pomc neurons is mediated via αand βmsh. human and animal studies have shown that a lack of mc4r leads to obesity, hyperphagia and insulin resistance (23, 24), and in obese persons βmsh mutations rather than α-msh (25). as mentioned above, melanocortin receptors are distinguished by their ability to bind ligand, and under normal physiological conditions, asp doesn`t have a role in the regulation of food intake since it antagonizes mc1r, but when asp is synthesized from dermal papilla cells in very high concentrations, it acts as an antagonist of mc4r (20). it is thought that agrp binds to neural mc3r and mc4r, and in genetically obese mice agrp is overexpressed, indicating that agrp has a physiological role in feeding behavior (20). no effect was observed on mc1r and mc2r. mc4r is specific since it binds both antagonists, asp and agrp, while mc3r binds agrp, and mc1r binds only asp (20). food availability – food quality (salt/sugar) as never before, the food-based dietary diversity strategy has been put into the focus of worldwide political attention and has social, cultural, economic and environmental benefits (26). although it is well known that dietary habits and health effects depend on ethnicity, gender, region and city (27), there is a widespread intention to improve world population health and individual health through diet (28). in the past, the concept of food quality was quite different compared to the present day, where an important role is given to modern marketing and seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 126 southeastern european medical journal, vol 1, 2017. sophisticated industrial production methods of processed food rich in added sugar and salt (29). in the everyday diet, the sugar content has tripled during the last 50 years, and there is critical concern about dietary fructose, which has various negative effects on body metabolism (30). it is even popular to compare its effects with those that result from alcohol intake. both metabolic processes are predominantly based on glycolysis, generate reactive oxygen species and result in a similar disease range (31). kitchen salt, sodium chloride, has a very important role in the food industry. the consumption of salt, even much more than is necessary for normal homeostatic process in the human organism, attenuates bitter taste and enhances taste in general. bakery products, a widely available and popular food category for every age group, are considered to be a major hidden salt source. in general, even though the word “addiction” has a negative connotation, much research suggests that consuming salty, sweet and fatty foods, for example fast food meals, can produce similar neurophysiological effects as in certain addictive drugs, leading to human obesity in the general population (32). that kind of processed food induces effects that are overwhelming to our brain-reward circuitry in a way natural food does not (30). innovative solutions need to be implemented by the food industry to replace sodium with more acceptable substances without customer dissatisfaction (33). the world health organization has taken aim at a 30% reduction in global salt intake by 2025 (34). croatia is an example of a country with an organized national program for the reduction of excessive salt intake, with the goal of helping to develop consciousness about the negative impacts of salt on hypertension (35). social interactions as extrinsic factor (mirror neurons) when discussing the physiological mechanisms related to food intake, we cannot avoid discussing what food means to human beings in addition to being simply “body fuel”. how does one distinguish between food as an elementary human need and food as part of social and emotional interaction? the external influence on individual food consumption is very complex; it depends on a variety of factors, and therefore some studies were overlap in the search for proper answers (36). human beings have a tendency to imitate the behavior of others including behavior related to food intake, a phenomenon that is known to exist in the animal world as well (37). through many psychological studies, it has been argued that the act of perceiving another person’s behavior creates a tendency to behave similarly oneself, which is called the “chameleon effect” (perceptionbehavior link) (38). in recent history, this and many similar correlations were explained by the discovery of “mirror” neurons —a distinct and intriguing group of neurons whose activities have been located in many different cortical areas, but were most extensively investigated in the ventral premotor region f5. they can transform specific sensory information into a motor format. moreover, we can say in a way that “motor” or “sensory” neurons have an impact on either execution or observation, and the “mirror” neurons have both (39). they have a huge role in learning during the first developmental periods of life (40). the described functions of mirror neurons can be used to explain the adoption of the first dietary habits from our parents and other close persons, because imitation is a fundamental method through which children learn from their parents, including choices of food and food quantity. according to that concept, obese children display an altered conscious perception of their own weight (41). in that sense, the mirror neurons may represent independent neurophysiological pathways to stimulate overeating and in that way increase calorie intake (37). furthermore, the perceived trend which includes a lack of physical activity combined with high calorie meals leads to a high bmi, and together they have been referred to as the “big two” contributors to obesity (41,42). as well as having a negative effect (43), peer relationships can also have positive influences on developing everyday habits (44). for instance, peer group physical activity is the strongest seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 127 southeastern european medical journal, vol 1, 2017. predictor for individual physical activity (40). it is not well explained, but stigmas also have a huge effect on food intake, whereas with some exceptions, negative characteristics are assigned to those people who consume large amounts of food (45). by applying a multidisciplinary approach and using sophisticated neuroimaging methods, more useful information could be obtained not merely about the relationship between mirror neurons and food intake habits, but also about human behavior in general. in conclusion, the pathophysiology of obesity includes the complicated interaction of the autonomic nervous system and hormonal system with the neuropeptide feedback loops from the gastrointestinal system to the hypothalamic centers that regulate energy homeostasis. moreover, it is significantly affected by human behavior, emotions and food intake. thus, obesity is a preventable risk factor for other cardiometabolic diseases. disclosures funding: no specific funding was received for this study. competing interests: none to declare. references 1. gurevich-panigrahi t, panigrahi s, wiechec e, los m. 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jm, lemon rn. what we know currently about mirror neurons. curr biol 2013;23(23):r1057–62. 41. salvy sj, de la haye k, bowker jc, hermans rc. influence of peers and friends on children’s and adolescent’s eating and activity behaviors. physiol behav 2012;106(3):369-78. 42. cdc, editor. centers for disease control and prevention. overweight and obesity: causes and consequences. 2009. 43. lumeng jc, hillman kh. eating in larger groups increases food consumption. arch dis child 2007;92(5):384–7. 44. priebe cs, spink ks. when in rome: descriptive norms and physical activity. psychol sport exerc 2011;12(2):93–8. 45. vartanian lr, herman cp, polivy j. consumption stereotypes and impression management: how you are what you eat. appetite 2007;48(3):265-77. seemedj 2018, vol 2, no. 1 hemochromatosis treatment by venipuncture 44 southeastern european medical journal, 2018; 2(1) hemochromatosis treatment by venipuncture through history 1 marija čuljak1 1 department of clinical chemistry, university hospital center sestre milosrdnice, zagreb, croatia corresponding author: marija čuljak, culjakmarija@gmail.com introduction there are various expressions used to describe the procedure of therapeutic blood sampling, venipuncture, phlebotomy, venesection and bloodletting. a widely used expression across the english-speaking world is “phlebotomy”, derived from the greek word “phleb”, which stands for “vein”, and “tómos” which means “incision”. phlebotomy is the process of opening a vein by incision in order to take blood for therapeutic treatment or diagnosis. one of the first people who recognized the importance of received: jan 3, 2018; revised version accepted: oct 12, 2018; published: november 27. 2018 keywords: venipuncture, phlebotomy, hemochromatosis, bloodletting veins and arteries was the chinese emperor known as the yellow emperor or huang-di (who ruled china in the 27th century bc). his famous remark was that “everything in the blood is under the control of the heart” (1). the ancient skill of bloodletting dates back to about three thousand years ago. ancient greeks, egyptians, the peoples of mesopotamia, the maya and the aztecs all knew the practice of blood-shedding, or bloodletting, as a treatment for diseases. this practice began with the egyptians 1000 years before christ and was abstract hemochromatosis is a hereditary disease caused by the mutation of genes responsible for regulating iron metabolism in the body. the mutation results in increased absorption of iron from food, which is then deposited in various organs and tissues. due to the excessive decomposition of iron, organs, most commonly liver, heart and pancreas are damaged. the standard therapeutic procedure for the treatment of hereditary hemochromatosis is phlebotomy or venipuncture, which removes excess iron from the blood. the therapeutic procedure is carried out until the iron level returns to the reference interval. ancient, three thousand years old bloodletting skills are still used to this day. it was considered that the bloodletting establishes a good balance of bodily fluids referred to as eucrasia. venipuncture was used to treat various diseases, with different amount of blood released, from half a liter to two liters, and sometimes even more. venipuncture was applied not only for the treatment of existing diseases, but also as a preventive measure. to date, the use of therapeutic venipuncture has remained the gold standard for the treatment of hereditary hemochromatosis. the aim of this paper is to present a historical review of venipuncture or phlebotomy as a therapeutic procedure for the treatment of hemochromatosis. (čuljak m. hemochromatosis treatment by venipuncture through history. seemedj 2018; 2(1); 4448) seemedj 2018, vol 2, no. 1 hemochromatosis treatment by venipuncture 45 southeastern european medical journal, 2018; 2(1) popular among doctors until the late 19th century. bloodletting was performed by shavers at the recommendation of a physician. barbershops that performed the procedure were recognized by a barber’s pole with red and white stripes. through history, bloodletting, especially with the help of leeches, was practiced as a treatment for existing diseases and as a method of prevention. still, not everything was bad in the treatment by bloodletting. medicinal leeches were used for improving circulation and blood flow during surgery. in some cultures, these treatments are still used today (2). according to hippocrates (460 bc 380 bc), a person’s personality (temperament) is determined by four body fluids (called “humors”): phlegm, yellow bile, black bile and blood. hippocrates defined health as a good mixture (eucrasia), and disease as a bad mixture of these juices (dyscrasia). he used clinical observations to recognize the symptoms of certain diseases. all four classical elements: fire, earth, water and air were considered present in the blood, and it was believed that the letting of blood would restore the patient’s general wellbeing. blood loss was considered beneficial because it was a way of balancing the fluids. hippocrates believed that the fluids had to be perfectly balanced in order for the individual to have good health. any imbalance was treated by bloodletting (3). in greece, galen of pergamon (129-200 ad), a prominent greek physician, surgeon and philosopher, first described that not only veins were filled with blood, but arteries as well, which were previously considered to be filled with air. he also believed in the humoral balance. he believed that blood should be released near the diseased part of the body. according to him, fever, termination of organ functions and headache are a result of excess blood, so the surgeon should tie the patient's hands to make the veins swell and become more pronounced in order to release a certain amount of blood (4). in hebrew teaching, specifically in the talmud, it is said that the release of blood should be carried out on certain days of the week and month. christianity adopts similar guidelines. islamic scholars also believe that the release of blood is very useful for the treatment of fevers. physical signs that required bloodletting were redness, swelling, skin warm to the touch, and any sign of pus. the treatment consisted of making numerous cuts on the body, and letting the blood flow into specially prepared receptacles. however, the treatment would be stopped, if the patient felt weak. interestingly, there is no data on the number of deaths during these procedures (2,3). hemochromatosis primary hemochromatosis is a hereditary disease caused by hfe gene mutation on chromosome 6. this is the most common autosomal recessive hereditary disease of the caucasian population in northern european countries, with prevalence in europe reported as 1: 300. it is equally represented amongst both male and female populations. sickness rarely occurs in the african and african american population, as well as in the asian population. men develop a clinical picture more often than women, about 10 times more often, the reason for this being the loss of iron in women during periods, birth and breastfeeding. hereditary hemochromatosis is divided into four subtypes, and only type 1 is clinically important. a total of 85-90% of patients with confirmed diagnosis of hemochromatosis are homozygotes for mutation c282y (y / y genotype), while about 5% are heterozygotes for mutations c282y, h63d and / or s65c. in individuals who did not demonstrate mutation c282y, and who are heterozygotes for s65c and / or h63d, the possibility of developing a clinical picture of inherited hemochromatosis is extremely low (5.6). diagnoses of hemochromatosis include laboratory diagnostics, radiological diagnostics and histological treatment. the clinical picture of hemochromatosis includes tiredness, hyperpigmentation (bronze skin color), loss of libido in men, diabetes, joint pain, cirrhosis of the liver and cardiomyopathy. seemedj 2018, vol 2, no. 1 hemochromatosis treatment by venipuncture 46 southeastern european medical journal, 2018; 2(1) laboratory diagnostics show elevated levels of serum iron and ferritin as well as increased transferrin saturation index. applying venipuncture in the treatment of hemochromatosis hemochromatosis was first described in paris in 1865 by armand trousseau, a french physician, who outlined the disease as a set of symptoms involving diabetes, skin pigmentation, and liver cirrhosis. trousseau did not name the disease (2). davis and arrowsmith are attributed with the first report on the treatment of hemochromatosis which showed the value of venipuncture. in their report, they state that repeated venipunctures can remove harmful iron. to confirm the diagnosis of hemochromatosis, they carried out a liver biopsy. the first patient was a 69-year-old woman. between march 1947 and february 1949, 40 liters of blood were drawn from this patient. according to their report, all patients treated by bloodletting showed a general improvement (7). in 1954, davey, foxell and kamp reported the case of a man at the age of 55 who was hospitalized in october 1950. the patient complained of abdominal pain that had lasted for 2 years, increased tiredness and gray and black pigmentation of the face, neck and upper arm. his liver was enlarged, spleen was not palpable, and free intra-abdominal fluid was found. he had not suffered from previous illnesses except for colon cancer surgery in 1936. he drank plenty of beer daily. the tested urine showed glucose in the urine. skin biopsy showed iron deposits, especially around the sweat glands. the patient was treated with repeated venesections. over the course of two years, 31 liters of blood were drawn from the patient. heavy anemia developed, although the patient showed subjective and objective improvement. the patient was last registered in 1954 when his symptoms were pain in the joints. a report was published, claiming that venesection is an effective hemochromatosis treatment method (8). in 1965, a group of authors (block, moore, wasi and haiby) investigated a cohort of patients with hemochromatosis. in their research, a liver biopsy was performed prior to venesection, the findings of which suggested that liver lesions arise due to abnormal absorption of iron despite a normal diet (9). in an article published in 1969, dr. r. williamson reported that over a period of 5 years, the mortality of patients treated with repeat venesection was 11%, while the mortality of untreated patients was more than 60% (10). sargent et al. report on two hypotheses regarding hereditary hemochromatosis. one hypothesis is that inherited hemochromatosis results in excessive absorption of iron from food consumed by the patient, which is the deposited in the organs, where it causes toxic organ damage. the second hypothesis is that hereditary hemochromatosis is actually a form of cirrhosis that is further complicated by excessive iron from the patient’s diet. in eight patients with hereditary hemochromatosis, iron levels were tested before, during and after therapy (venipuncture). in all cases, serum iron levels after therapy were within normal limits, as were other blood indices. this is in favor of the hypothesis that inherited hemochromatosis is a disorder in the mechanism responsible for controlling iron absorption from food (11). untreated hemochromatosis can result in death due to cardiac complications. heart damage is manifested as cardiomyopathy, arrhythmia and heart failure. a period of 12 months is required to restore normal liver function and up to 30 months to fully recover from angina. continuous and controlled therapy (venipuncture) is needed to restore normal parameters (12). an early diagnosis of hereditary hemochromatosis has a tremendous significance in preventing the development of diabetes mellitus as well as hepatocellular carcinoma. niederau et al. claim that patients who received timely therapy prior to the onset of cirrhosis did not develop hepatocellular carcinoma later. early treatment with venipuncture prevents the formation of cirrhosis and attention should therefore be focused on early diagnosis of hereditary hemochromatosis (13). seemedj 2018, vol 2, no. 1 hemochromatosis treatment by venipuncture 47 southeastern european medical journal, 2018; 2(1) a research conducted by a group of authors in norway shows that the treating of hereditary hemochromatosis by venipuncture leads to a change in the level of some trace elements, including increased absorption of toxic elements such as copper. (14). conclusion therapeutic venipuncture or phlebotomy is the golden standard for the hemochromatosis therapy. in the procedure, 500 ml of blood containing 200 to 250 mg of iron is extracted. standard procedures prescribe periodic venipuncture, approximately once a week, with the letting of 400-500 ml of blood, during the first 3 years, followed by 400-500 ml every 1-3 months. venipuncture is carried out until the levels of iron and ferritin drop to the lower limit of the normal reference interval. venipuncture can be performed on younger patients 2-3 times a week. the aim is to achieve ferritin values below 50 ng / ml and a ferritin saturation index under 50% in serum. most patients can tolerate quite well a week of undergoing the procedure, which should be performed until hematocrit falls below 37%. to monitor the therapeutic response, it is recommended to check serum saturation and serum ferritin levels every 2 to 3 months (15). treatment of primary hemochromatosis by venipuncture is a safe and effective way of removing blood in order to reduce iron levels. treatment is a lengthy process, usually lasting several years until iron values return to normal. venipuncture improves the overall condition of the patient, alleviates the symptoms of fatigue, pain, and skin pigmentation. regular therapy prevents liver complications, heart disease and diabetes (16). despite the advances in technology, bloodletting as a therapeutic procedure has survived to this day as a controlled, supervised, safe and efficient treatment option. in this procedure, it is important to follow all the guidelines for proper blood drawing. the role of medical laboratory personnel in diagnosing and monitoring primary hemochromatosis is vital. laboratory tests allow the physician to make a proper diagnosis and evaluate the overall medical condition during the course of disease monitoring acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. history of phlebotomy. available at: https://phlebotomycoach.com/resources/ history-of-phlebotomy, accessed october 25th, 2017. 2. depalma rg, hayes vw, zacharski lr. bloodletting: past and present. j am coll surg 2007;205(1):132-144. 3. colović n, leković d, gotić m. treatment by bloodletting in the past and present. srp arh celok lek 2016;144:240-248. 4. long history of phlebotomy-bloodletting. available at: https://hubpages.com/education/longhistory-of-phlebotomy, accessed october 25th, 2017. 5. ulvik rj. hereditary haemochromatosis through 150 years. tidsskr nor laegeforen 2016; 136:2017-2021. 6. topić e, primorac d, janković s. medicinsko biokemijska dijagnostika u kliničkoj praksi. medicinska naklada, zagreb, 2004 (in croatian). 7. davis wd jr, arrowsmith wr. the effect of repeated bleeding in hemochromatosis. j lab clin med. 1950;36:814-815. 8. davey da, foxell aw, kemp ta. treatment of haemochromatosis by repeated venesection. br med j 1954;2:1511-4. 9. block m, moore g, wasi p, haiby g. histogenesis of the hepatic lesion in primary hemochromatosis: with consideration of the pseudo-iron deficient state produced by phlebotomies. am j pathol 1965;47:89-123. seemedj 2018, vol 2, no. 1 hemochromatosis treatment by venipuncture 48 southeastern european medical journal, 2018; 2(1) 10. crosby wh. hemochromatosis and venesection. br med j 1969;4:109-10. 11. sargent t, saito h, winchell hs. iron absorption in hemochromatosis before and after phlebotomy therapy. j nucl med 1971;12:660-7. 12. feely j, counihan tb. haemochromatosis presenting as angina and responding to venesection. br med j 1977;2:681-8. 13. finlayson nd. hereditary (primary) haemochromatosis. bmj 1990;301:350-1. 14. crosby wh. a history of phlebotomy therapy for hemochromatosis. am j med sci 1991;301:28-31. 15. bolann bj, distante s, mørkrid l, ulvik rj. bloodletting therapy in hemochromatosis: does it affect trace element homeostasis? j trace elem med biol. 2015;31:225-9. 16. cook ls. therapeutic phlebotomy: a review of diagnoses and treatment considerations. j infus nurs 2010;33:81-8. seemedj 2019, vol 3, no. 2 open approach in rhinoplasty 56 southeastern european medical journal, 2019; 3(2) review article open approach in rhinoplasty 1 vjeran bogović 1, stjepan grga milanković 1, željko zubčić ¹, andrijana včeva ¹, martina mihalj ², josip maleš ¹, željko kotromanović ¹, mirjana grebenar ¹, tihana mendeš 1, hrvoje mihalj 1 1 department of otorhinolaryngology, head and neck surgery, clinical hospital centre osijek, croatia 2 department of physiology and immunology, faculty of medicine, osijek, croatia corresponding author: hrvoje mihalj, mihalj.hrvoje@kbo.hr received: may 24, 2019; revised version accepted: sep 19, 2019; published: dec 16, 2019 keywords: rhinoplasty, diagnosis, follow up abstract the aim of this study was to present the advantages and disadvantages of open approach in rhinoplasty. in it, we also present the development of this technique in croatia and examine its application in the last five years at the department of otorhinolaryngology and head and neck surgery of the clinical hospital centre osijek. retrospectively, from january 2008 to august 2012, 400 patients with septal deviation and/or deformities of the nasal pyramid who underwent open rhinoplasty at the department of otorhinolaryngology, head and neck surgery of the clinical hospital centre of osijek, croatia were identified. the clinical diagnosis was based on a detailed medical history of the patient and nasal endoscopy. the patients were photographed in six projections before and after the surgery, and followed up on two occasions. (bogović v, milanković sg, zubčić ž, včeva a, mihalj m, maleš j, kotromanović ž, grebenar m, mendeš t, mihalj h. open approach in rhinoplasty. seemedj 2019; 3(2); 56-62) seemedj 2019, vol 3, no. 2 open approach in rhinoplasty 57 southeastern european medical journal, 2019; 3(2) introduction rhinoplasty is a procedure in plastic surgery of the nose which solves functional and aesthetic problems of nasal soft tissue. rhinoplasty includes repositioning of nasal cartilage and bone as well as of soft tissue structures for the purpose of improving the function and aesthetic appearance of the nose (1). it is often necessary to do both rhinoplasty and septoplasty (surgery of the nasal septum which facilitates breathing) at the same time. such combined surgery is called rhinoseptoplasty. nasal deformities affect the aesthetics of the face, and rhinoplasty affects the individual functionally, aesthetically, artistically and psychologically. rhinoplasty is one of the most complex and most challenging interventions in aesthetic surgery, where precision is down to a fraction of a millimetre. anatomy of the nose is very complex, with challenging bone and cartilage structure, as well as variable thickness of the skin, with the thickest skin found on the apex. it is necessary to precisely determine how a certain procedure will affect the appearance of the nose (2). history of medicine notes that the first successful aesthetic surgery of the nose was performed by the brancas (father and son) in the 15th century, in the italian province of catania (3). at the beginning, rhinoplasty was considered to be a surgical part of psychotherapy, which is confirmed by the fact that at the start of the 20th century, depression was one of the indications for rhinoplasty. the father of modern techniques of rhinoplasty is jacques joseph, who lay the foundation of the surgical technique in 1928 (4). at the same time, in 1921, an otolaryngologist from budapest named aurel rethy developed a new surgical technique, the so-called open approach or decortication. rethy’s technique was first modified by prof. ante šercer, and his modified technique is still being applied all over the world today. professor ante šercer was born in požega, completed his studies in graz and prague, and worked at the two biggest clinical hospital centres (chc) after world war ii: “šalata” (chc zagreb) and chc sestre milosrdnice. he is recognized as the greatest croatian expert in otorhinolaryngology. his student, prof. ivo padovan, presented a new open approach technique (so-called decortication) in 1970 at the first international symposium of the american academy of facial plastic and reconstructive surgery in new york, showing the results of the procedure conducted on 400 patients, who were operated at the department of otorhinolaryngology of the chc “sestre milosrdnice” (5). according to the claims of prof. gunther, a famous american plastic surgeon, who is the organizer of one of the most popular international symposia for plastic and reconstructive head and neck surgery in dallas, that method was not accepted in the usa until the 1970’s. today, a lot of ent specialists and specialists and subspecialists of plastic and reconstructive head and neck surgery use decortication all over the world. as we can see from the available data, 98% of patients are operated on by surgeons using this approach at the clinical hospital in dallas. the respected prof. toriumi from chicago uses the open approach for more than 85% of all rhinoplasty procedures. in the usa, surgeons perform over 100 000 aesthetic rhinoplasties annually (6). the nose is located at the centre of the face; it is also the most protruding part of the face, which is why it is the most exposed part of the face for trauma. it can be changed as a result of trauma, genetic factors or disease. nasal trauma is the most common cause of aesthetic deformities of the septum and of the external part of the nose. aesthetic nose correction is the most challenging surgery in aesthetic surgery. the spectrum of deformities of the external part of nose is varied and because of that, the approach and planning of surgical procedure must be done on a case-by-case basis. typical nose deformities include: dorsal hump, saddle nose, crooked nose, overprojected nose, wide nose and combinations of the above. the goal of rhinoplasty is to create the nose shape that would best fit the face, because every face is different, and the nose needs to be adjusted seemedj 2019, vol 3, no. 2 open approach in rhinoplasty 58 southeastern european medical journal, 2019; 3(2) accordingly. rhinoplasty may be primary (correction for the first time) or secondary (reoperation). there are two main approaches to rhinoplasty: intranasal and open (decortication). deformities may be located in the bone and/or cartilage. in the open approach, a small incision is performed on the columella and along the front edge of alar cartilage, and in that way, deformities may be corrected . open approach is indicated in severe deformity of the apex, or for noses that have been operated multiple times or have suffered trauma multiple times, since they cannot be operated on using the intranasal approach. in the open approach, a small scar is left in the shape of the letter v at the columella (the fleshy part that divides the nostrils). skin haematoma of the apex lasts longer than when the intranasal technique is used, which depends mostly on the type of skin on the apex. breathing normally through the nose should never be sacrificed to the achieve the desired aesthetic nose shape (7). patients and methods this retrospective analysis was made at the department of otorhinolaryngology and head and neck surgery of the clinical hospital centre osijek. a total of 400 patients with functional and/or aesthetic nose surgeries performed in the period from january 2008 to august 2012 were included (table 1). table 1. functional and aesthetic rhinoplasty performed at the department of otorhinolaryngology and head and neck surgery of the clinical hospital centre osijek from 2008 to 2012. type of surgery 2008 2009 2010 2011 8/2012 total septoplasty 44 56 57 67 55 279 rhynoseptoplasty (open and intranasal approach) 12 17 18 32 21 100 (28, 72) reconstruction of nasal valve 0 4 8 5 4 21 total 400 study design before surgery, after taking the patient’s anamnesis, the patient was photographed and analysed from six different angles. attention was drawn to the nasolabial angle, which is 90-110 degrees in women, and 80 to 90 degrees in men, while the nasofrontal angle is generally about 150 degrees in both sexes (figure 1). seemedj 2019, vol 3, no. 2 open approach in rhinoplasty 59 southeastern european medical journal, 2019; 3(2) figure 1. preoperative photo after thorough anamnesis was taken, the preoperative patient was photographed and analysed from six different angles (figure 1a) (5). attention is drawn to the nasolabial angle, which is 90-110 degrees in women, and 80 to 90 degrees in men, while the nasofrontal angle is generally about 150 degrees in both sexes. figure 1b shows deviations from the median plane and clearance of the nose and preoperative planning of the operation. functional and/or aesthetic nose surgeries performed were septoplasty, rhinoseptoplasty (open or intranasal approach) and reconstruction of the nasal valve. after the surgery was completed, the results were monitored and documented by repeated photographing from the same six angles (figure 2). seemedj 2019, vol 3, no. 2 open approach in rhinoplasty 60 southeastern european medical journal, 2019; 3(2) figure 2. postoperative photo the results of the operation are monitored and documented by repeated photographing from six angles. figure 2 shows the same patient after rhinoseptoplasty was performed; the increase in the nasolabial angle is noticeable, as is the corrected nasal dorsum. results we noted 279 septoplasties, 100 rhinoseptoplasties and 21 reconstructions of the nasal valve (table 1). from the presented data, it is evident that the number of surgical interventions at the annual level is constantly increasing, which is in line with the increase in the number of ent surgeons employed at our hospital, who were educated at other hospitals in croatia, as well as in world-renowned centres for plastic reconstructive surgery. discussion in addition to the functional dimension, the nose has an essential aesthetic dimension. in functional terms, the nose is the initial and extremely important part of the respiratory system. apart from the “air conditioning” the inhaled air, the nose also contains nerve endings that are responsible for distinguishing odours (5). generally, the appearance, i.e. the anatomic material, reflects the function of the breathing dynamics. it is therefore clear that any plastic surgery performed on the nose is also a functional surgery. along with the aforementioned “technical” aspect, the appearance of the nose is affected by race, ethnic characteristics and individual heritage. within each race, there are typical characteristics. in the black race, for example, the nose is wider than the nose in the white race; it is lower in the bony part, with a predominance of wide noses, and the columella is shorter, with horizontal nostrils. in the white race, we differentiate the greek, the latin, the galician, the germanic nose, etc. (8). finally, the appearance of the nose can be affected by the surgeon. it is not unusual that, apart from correcting individual characteristics, members of individual races or ethnic groups want to reshape the basic characteristics of their nose; for example, in asia, people tend to strive to “westernize” by changing the east asian elements of their physiognomy. seemedj 2019, vol 3, no. 2 open approach in rhinoplasty 61 southeastern european medical journal, 2019; 3(2) surgery requires four fundamental criteria – three objective and one subjective: balanced form, good function, correct proportions, and desired appearance. the common notion that the nose changes the profile is only partially correct because rhinoplasty affects the shape of all projections. if the surgery meets the objective requirements, the surgeon may be satisfied, but for complete success, the patient must also be satisfied. therefore, it is necessary that the surgeon, prior to performing the surgery, has an interview with the patient to find out which anatomical specifics the patient is not satisfied with and what the patient’s particular aesthetic expectations are regarding the appearance of the nose. this is important because patients sometimes have unrealistic expectations of surgery or motivations that are not in fact related to the appearance of the nose, but are caused by other social and psychological circumstances that they project on their external appearance (9). complications of rhinoplasty are relatively rare (10). possible complications include: nosebleeds, swelling of the nose and the surrounding soft tissue of the eyes and cheeks (especially in “open” rhinoplasty, lasting up to one year), bleeding, infections, hypertrophic scars, redeformation of the nose (due to scars and poor surgical techniques). some patients can develop difficulty breathing through the nose following rhinoplasty, despite a successful aesthetic outcome of the operation. even though the inside of the nose generally allows for good passage of air, rhinometric measurement of the airflow should measure the nasal discharge insufficiency. this is damage to the fine mechanism responsible for normal airflow in the throat and normal discharge, and it may be congenital or acquired. treatment of this condition is very difficult and unpredictable, so it is best if it is prevented by proper surgical technique. decisions regarding nose surgery should be preceded by detailed discussion and review of the situation. on that occasion, the doctor explains the nature of the problem to the patient and the realistic possibility of its correction. after rhinoplasty, a patch, plaster splint or plastic material is used to keep the shape and position of the nose for the first 7-10 days until it is healed. nose and face swelling is individual, depending on the severity of the surgery itself and the response of the patient’s tissue. a return to daily activities is possible after approximately two weeks, depending on the individual situation. strenuous physical activity should be avoided over the next two months. it is important to understand that the recovery process after rhinoplasty takes from six months to a year, until the nose reaches its final appearance. the final judgment on the appearance and function of the nose is made by the patient and the surgeon only after five years of patient monitoring, which is particularly the case in the west (10). bleeding after the removal of the tampon is the most commonly observed complication; it is usually weak and stops after a few minutes. in some cases, the tampon is replaced until the next day. sweating is not a common complication. the nose has very good blood supply, which provides a strong immune defence against pathogens, and today’s medical institutions also follow stringent protocols regarding sterilization and instrument handling. unsatisfactory aesthetic effect of the surgery is considered to be one of the late-stage complications of rhinoplasty (11). in some cases, the result of the surgery differs from what the patient expected, and this usually occurs when the desired look does not match the result achieved. to prevent this, realistically expected results are discussed prior to surgery, as well as after surgery. when images before and after the surgery are compared to help patients see the improvement in their appearance, most patients are satisfied. repetitive rhinoplasty, also known as secondary rhinoplasty (12), is rhinoplasty performed to correct inadequate results of the previous rhinoplasty. there are two reasons for performing secondary rhinoplasty. patients often seek secondary rhinoplasty to correct a cosmetic nasal deformity. nose fracture may not have been sufficiently reduced or was done too low (13). the overprojected nose may not be adequately resolved, or it may have been overly resolved. it may look squashed; it may look like a bell pepper or like a boxer’s nose. there are seemedj 2019, vol 3, no. 2 open approach in rhinoplasty 62 southeastern european medical journal, 2019; 3(2) many ways in which the results of the previous rhinoplasty can be aesthetically unattractive to the patient. another reason is the functionality. in the first case, rhinoplasty may be done to alleviate breathing difficulties, and the result may be unsatisfactory. in the second case, the first rhinoplasty may be performed for cosmetic reasons, but may also endanger the normal physiological mechanism that involves inhaling or exhaling, making it difficult to breathe. secondary rhinoplasty is technically challenging and numerous procedures have to be done by open rhinoplasty. in conclusion, advancements in rhinoplasty techniques, e.g. stabilizing the cartilage graft and open access, now provide satisfactory results in secondary rhinoplasty that had not been possible in the past (14). acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. rohrich r, ahmad j. rhinoplasty. plast reconstr surg 2011; 128(2):49-73. 2. vilar-sancho b. rhinoseptoplasty. aesthetic plast surg 1984; 8(2):61-65. 3. greco m, ciriaco ag, vonella m, vitagliano t. the primacy of the vianeo family in the invention of nasal reconstruction technique. ann plast surg 2010; 64:702-5. 4. bhattacharya s. jacques joseph: father of modern aesthetic surgery. indian j plast surg. 2008;41(suppl): s3-s8. 5. šercer a. otorinolaringologija 2, klinika. zagreb: jlz, 1965, 356-357. 6. gunter jp, rohrich rj, adams, wp (eds.). dallas rhinoplasty – nasal surgery by the masters, 2nd edn., st. louis: quality medical publishing inc; 2007 7. sercer a, mundich k. plastische operationen an der nase und an der ohrmuschel, stuttgart: georg thieme verlag, 1962 8. leong s, eccles r. a systematic review of the nasal index and the significance of the shape and size of the nose in rhinology. clin otolaryngol 2009; 34(3):191-8. doi: 10.1111/j.17494486.2009.01905.x. 9. naraghi m, atari m. development and validation of the expectations of aesthetic rhinoplasty scale. arch plastic surgery 2016; 43(4):365-370. 10. cochran cs, landecker a. prevention and management of rhinoplasty complications. plast reconstr surg 2008; 122:60-67. 11. rettinger g. risks and complications in rhinoplasty. gms curr top otorhinolaryngol head neck surg. 2007;6:doc08. 12. gruber rp, noland s, belek ka. discussion: what motivates secondary rhinoplasty? a study of 150 consecutive patients. plast reconstr surg 2012; 130:679-80. 13. philpott cm, clark a, mckiernan dc. function or cosmesis--what is the predominant concern in patients with nasal trauma presenting for rhinoplasty? eplasty 2009;9:e11. daniel r. secondary rhinoplasty following open rhinoplasty. plast reconstr surg 1995; 96(7):1539-1546. seemedj 2017, vol 1, no. 2 agnor counts in differential diagnosis of parathyroid adenoma and hyperplasia… 19 southeastern european medical journal, 2017; 1(2) agnor counts in differential diagnosis of parathyroid adenoma and hyperplasia in preoperative cytologic smears 1 ljubica fustar preradovic1, davorin danic2, ika kardum-skelin3, bozena sarcevic4, ana danic hadzibegovic5 1 department of pathology and cytology, dr. josip bencevic general hospital, slavonski brod, croatia 2 department of ent and head and neck surgery, dr. josip bencevic general hospital slavonski brod, croatia and faculty of medicine osijek and faculty of dental medicine and health, josip juraj strossmayer university of osijek, croatia 3 department of cytology and cytogenetics, “merkur” university hospital, zagreb, croatia school of medicine university of zagreb, croatia 4 department of pathology, university hospital for tumors, clinical center “sestre milosrdnice”, zagreb, croatia and school of medicine university of zagreb, croatia 5 clinical department for ent and head and neck surgery, clinical hospital center zagreb, zagreb, croatia and faculty of medicine, josip juraj strossmayer university of osijek, croatia corresponding author: ljubica fustar preradovic – ljubica.fustar.preradovic@sb.t-com.hr received: july 14, 2017; revised version accepted: november 24, 2017; published: november 24. 2017 keywords: agnors, parathyroid adenoma, parathyroid hyperplasia abstract objective. minimally invasive surgery is the method of choice in the management of hyperparathyroidism caused by parathyroid adenoma, whereas in case of parathyroid hyperplasia a radical operative procedure is necessary to prevent recurrence of the disease. the aim of the study was to investigate morphological and cytochemical parameters differentiating parathyroid adenoma from parathyroid hyperplasia in cytologic smears in preoperative work-up of patients with hyperparathyroidism. methods. fifty parathyroid cytologic smears, preoperatively obtained by ultrasound-guided aspiration biopsy, were analyzed. fifty parathyroid cell nuclei per smear were analyzed, and the number of nucleolar organizer region (agnor) was determined using sform software (vamstec, zagreb). the results obtained were compared with histopathology findings. results. the values of nuclear size parameters obtained by morphometric measurement revealed cells with larger nuclei and greater nuclear size diversity to be found in parathyroid adenoma, thus enabling differentiation of parathyroid hyperplasia from parathyroid adenoma in many cases. however, due to overlapping of borderline values, an attempt was made to reduce the possibility of error by determining agnor count and structure. the results obtained showed that neither agnor count, nor agnor classification into individual agnor, agnor clusters and annular agnor, had any role in differentiating parathyroid hyperplasia from parathyroid adenoma. seemedj 2017, vol 1, no. 2 agnor counts in differential diagnosis of parathyroid adenoma and hyperplasia… 20 southeastern european medical journal, 2017; 1(2) introduction parathyroid adenoma is mostly found as a solitary tumor, therefore minimally invasive surgery has been accepted as the method of choice in the management of hyperparathyroidism caused by parathyroid adenoma. in contrast, parathyroid hyperplasia usually develops as a secondary lesion characterized by diffuse or nodular hyperplasia of one or more parathyroid glands. recurrence is known to occur in patients with parathyroid hyperplasia even after radical and properly performed surgery that required bilateral neck exploration, and such cases have been described in the literature, pointing to the importance of preoperative differentiation of parathyroid hyperplasia and parathyroid adenoma (1,2,3). nucleolar organizer regions (agnor) are segments located on the short arm of acrocentric chromosomes in which the rrna gene is located during the interphase and mitosis. these regions can be shown cytochemically by silver staining using the method of identifying non-histone, argyrophilic proteins connected with the sites of protein rrna transcription (agnor). agnors play a significant role in nucleic acid transcription to proteins, thus their number may serve as an indicator of cell proliferation and tumor growth (4-7). the present study was focused on identification of new morphological parameters for differentiation of parathyroid hyperplasia from parathyroid adenoma in preoperative cytologic smears by determining the number and structure of agnor per nucleus. material and methods fifty parathyroid gland smears obtained by ultrasound-guided fine-needle aspiration biopsy (us-fnab) were included in the study. the histopathologic diagnosis was unknown. study material was obtained by ultrasoundguided fine-needle aspiration biopsy (ug-fna) performed by a clinical cytologist (interventional cytologist) (8), who also performed the cytomorphological analysis of the material obtained. each patient signed an informed consent form, while the study protocol was approved by the hospital ethics committee and by the zagreb school of medicine ethics committee. us examinations were performed on an acuson x300 (siemens, erlangen, germany) us device with 8.9 mhz and 11 mhz superficial tissue probes. ug-fna was carried out by the free-hand technique (9). all smears were air dried and stained by the standard maygrünwald-giemsa (mgg) method (10) and cytochemical method of selective silver staining of agnor. for the cytochemical method, slides previously stained by the standard mgg method were used for allowing selection of appropriate slides with an adequate number of preserved cells. the selected slides were immersed in a solution containing 1 part of 2% gelatin solution in 1% formic acid and two parts of 50% aqueous silver nitrate solution, and then left to stay in a dark chamber at room temperature for 45 minutes. then the slides were washed with distilled water and air dried (12). upon silver impregnation, the agnor are seen as darkbrown spots in the nucleus (figure. 1). each slide was examined under olympus bx 50 microscope connected to a computer image analyzer, initially at low magnification to select a representative area; then the selected area was systematically explored under immersion objective (magnification x1000) (11,14). in the present study, agnor analysis was performed by dividing them into three types: 1) homogeneous type (where dilutions were not even visible at magnification); 2) inhomogeneous conclusion. study results showed that agnor count and structure cannot help in determining more clearly the border between parathyroid adenoma and hyperplasia in cytologic smears. (fustar preradovic lj, danic d, kardum-skelin i, sarcevic b, danic hadzibegovic a. agnor counts in differential diagnosis of parathyroid adenoma and hyperplasia in preoperative cytologic smears. seemedj 2017; 1(2); 19-26) seemedj 2017, vol 1, no. 2 agnor counts in differential diagnosis of parathyroid adenoma and hyperplasia… 21 southeastern european medical journal, 2017; 1(2) type (irregular agnor dilutions visible at x1000 and higher computer magnifications); and 3) annular agnor (annular configuration visible at standard magnification) (11,12). intranuclear agnor number was determined for each agnor type. all patients were eventually treated surgically. upon completion of testing, patients were divided into two groups based on histopathologic findings: parathyroid hyperplasia and parathyroid adenoma. the group of parathyroid hyperplasia included 36 (72%) patients and the group of parathyroid adenoma included 14 (28%) patients. the basic descriptive parameters (minimal/maximal value and arithmetic mean) and variability measures (coefficient of variation and standard deviation) were calculated for each continuous variable. the pattern of distribution was assessed for each continuous variable. determination was made of the mean value as a measure of central tendency and a central value based on the value size, as well as standard deviation (sd) as a measure of deviation of arithmetic means of samples from one population from arithmetic means of the other population. the correlation of continuous variables in the two groups was analyzed by the mann-whitney test as the most sensitive nonparametric test, along with the rank-sum calculation (13). results individual agnor count per nucleus was lower in the group of patients with parathyroid adenoma than in those with parathyroid hyperplasia, but the difference did not reach statistical significance (p=0.167) (table 1). the mean number of agnor clusters was comparable in the two groups (p=0.763) (table 2). the mean number of individual agnor and agnor clusters, the number of agnor clusters and annular agnor, the number of individual and annular agnor, and the number of all agnor, were greater in the group of patients with parathyroid hyperplasia than in those with parathyroid adenoma; however, the difference was not statistically significant (p=0.257, p=0.846, p=0.151, p=0.158) (table 3) (fig. 2). discussion preoperative morphological finding of parathyroid hyperplasia or parathyroid adenoma is fundamental for further treatment of patients with hyperparathyroidism. optimal patient management requires the highest possible differentiation between patients with parathyroid hyperplasia and those with parathyroid adenoma because minimally invasive surgery has been accepted as the method of choice in the management of hyperparathyroidism due to parathyroid adenoma, whereas recurrence is quite frequent in patients with parathyroid hyperplasia, thus figure 1. pg cells – agnor (silver nitrate, x1000) figure 2. mean value and confidence interval for agnor numbers hpl ad 0,00 0,10 0,20 0,30 0,40 b ro j n a k u p in a i p rs te n a s ti h a g n o r -a hpl ad 1,00 2,00 3,00 4,00 b ro j s v ih a g n o r -a hpl ad 1,00 2,00 3,00 4,00 b ro j p o je d in a č n ih i n a k u p in a a g n o r -a hpl ad 1,00 2,00 3,00 4,00 b ro j p o je d in a č n ih i p rs te n a s ti h a g n o r -a seemedj 2017, vol 1, no. 2 agnor counts in differential diagnosis of parathyroid adenoma and hyperplasia… 22 southeastern european medical journal, 2017; 1(2) demanding a radical surgical approach (1-3). on table 1. mean values of individual agnor parameters according to patient groups parameter hyperplasia adenoma p† mean sd* min max mean sd* min max individual agnor count 2.48 0.63 1.12 4.90 2.25 0.51 1.44 3.18 0.167 *standard deviation; †mann-whitney test table 2. median of agnor cluster number according to patient groups parameter hyperplasia adenoma p† median 25%-75%* min max median 25%-75%* min max number of agnor clusters 1.00 1 1 0.00 1.25 1.00 1 1 0.00 1.14 0.763 * interquartile range; †mann-whitney test table 3. median of agnor count according to patient groups parameter hyperplasia adenoma p† median 25%-75%* min max median 25%-75%* min max number of individual agnor and agnor clusters 2.48 2.16-2.74 1.12 5.20 2.36 1.86-2.62 1.48 3.34 0.257 number of agnor clusters and annular agnor 0.08 0.02-0.16 0.00 1.00 0.05 0.02-0.37 0.00 1.00 0.846 number of individual and annular agnor 2.42 2.16-2.69 1.12 4.90 2.29 1.85-2.44 1.44 3.18 0.151 number of all agnor 2.48 2.16-2.74 1.12 5.20 2.31 1.86-2.54 1.48 3.34 0.158 * interquartile range; †mann-whitney test seemedj 2017, vol 1, no. 2 agnor counts in differential diagnosis of parathyroid adenoma and hyperplasia… 23 southeastern european medical journal, 2017; 1(2) the other hand, subtotal resection of parathyroid gland or complete excision with partial parathyroid autotransplantation is performed on surgical removal of enlarged parathyroid glands in order to prevent consequential hypoparathyroidism; therefore, parathyroid tumor should be differentiated preoperatively from hyperplastic tissue. on morphometric measurements performed by the system of image analysis, the subjective investigator's error is reduced by use of quantitative parameters. in our earlier research, subsequent correlation with histopathologic findings revealed higher values of nuclear area, circumference, minimal radius, convexity and width in the group of patients with parathyroid hyperplasia compared with the group of patients with parathyroid adenoma (14). when extrapolated to cell morphology in cytologic smear, cells with greater nuclei were found in the group with hyperplasia in relation to those found in the group with adenoma. in the group with parathyroid hyperplasia, higher values of standard deviation were recorded for each of these parameters compared to parathyroid adenoma; applied to cell morphology in cytologic smear, it indicates greater heterogeneity and size diversity in hyperplasia as compared with adenoma (14). however, as the borderline values of the parameters analyzed showed an occasional overlap, we tried to more clearly determine the border between adenoma and hyperplasia in cytologic smears using additionally the cytochemical method and determination of nuclear agnor count. as the agnor count correlates with the level of dna transcription and degree of cell proliferation, the features of nuclear organization provide a way to simply distinguish resting cells from those involved in the cell cycle (4-7,15,16). black spots can be visualized in all cells of parathyroid epithelium by staining the nucleoli and other structures of nucleolar organization. agnor clusters were only detected in proliferating cells of various tissues, whereas individual spots were found in mature, nonproliferating cells (17-20). these findings point to physiological differences between individual agnor and agnor clusters, thus justifying their separate evaluation. there are literature reports on the studies where agnor count per nucleus was determined in postoperative parathyroid histopathologic smears and significant difference was found between parathyroid carcinoma and benign parathyroid lesions, while no such difference in agnor count per nucleus was recorded between parathyroid adenoma and parathyroid hyperplasia (21,22). boquist reports on the comparable agnor count per nucleus in parathyroid adenoma and liver adenoma (2.6 and 2.3 agnor per nucleus, respectively), breast fibroadenoma and thyroid follicular adenoma (23). mourad et al. have introduced the mean agnor (magnor) count per nucleus, which correlates with ploidy (aneuploid cells have ≥2.4 magnor per cell and diploid cells <2.4 magnor per cell (24). in the present study, agnor analysis was performed by dividing them into three types: homogeneous, inhomogeneous and annular agnor (8). as agnor clusters are only found in proliferating cells, the number of agnor clusters is understandably low in adenoma and hyperplasia; instead, they are only found in individual cells. in our study, annular agnor were only detected in several individual nuclei. irrespective of their number, all spots were enumerated and their role in differentiating parathyroid hyperplasia from parathyroid adenoma was assessed upon statistical data processing. the mean number of all agnor per nucleus was greater in parathyroid hyperplasia compared to parathyroid adenoma (2.48 vs. 2.31); the difference is not statistically significant (p=0.158) (table 3) and is consistent with the values reported by boquist in histopathologic material (23). statistical data processing indicated the agnor classification according to their proliferation activity into individual agnor, agnor clusters and annular agnor to have no value in differentiating parathyroid hyperplasia and parathyroid adenoma. these cells are lowproliferating cells predominated by homogeneous agnor, while agnor clusters and annular agnor are few and found in comparable number in both parathyroid hyperplasia and parathyroid adenoma. cytologic smears are considered more appropriate for agnor determination than histologic sections because the spots are more seemedj 2017, vol 1, no. 2 agnor counts in differential diagnosis of parathyroid adenoma and hyperplasia… 24 southeastern european medical journal, 2017; 1(2) easily enumerated and individual spots are better visualized within the nucleoli in cytologic smears. in the latter, whole cells are seen, thus the number of spots is expectedly higher than in histologic sections. yet, even materials that are so different can be compared if the ratio of agnor count and nucleus area is determined. this is of great value because there are few reports on studies performed on preoperatively obtained cytologic fnab smears, thus obviating possible errors caused by different sample processing (25-27). our own observations and subjective assessment that differences in nuclear size are substantial features to differentiate parathyroid adenoma and parathyroid hyperplasia in cytologic smear, and that these features can help differentiate these two entities in preoperative cytologic us-fnab smears, were quantitatively confirmed by use of objective morphometric measurement and statistical analysis of the data obtained. our results still point to justifiable utilization of cytodiagnosis, along with morphological and morphometric methods in the preoperative identification of parathyroid tumors. particular nuclear morphometric parameters may influence the selection of patients as potential candidates for minimally invasive or radical surgical procedure (14). however, the borderline values of the study parameters may occasionally overlap, and quantitative determination of agnor per nucleus in preoperative cytologic smears cannot help in differentiating parathyroid adenoma from parathyroid hyperplasia, thus it cannot be used as a diagnostic criterion, which is consistent with literature data obtained on postoperative histopathology material (23,28,29). acknowledgement we would like to thank drazen svagelj, md, phd for assistance and for comments that greatly improved the manuscript. funding: no specific funding was received for this study. competing interests: none to declare. references 1. laird am, libutti sk. minimally invasive parathyroidectomy versus bilateral neck exploration for primary hyperparathyroidism. surg oncol clin n am 2016;25(1):103-18. 2. rosato l, raffaelli m, bellantone r, pontecorvi a, avenia n, boniardi m, brandi ml, cetani f, chiofalo mg, conzo g, de palma m, gasparri g, giordano a, innaro n, leopaldi e, mariani g, marcocci c, marini p, miccoli p, nasi p, pacini f, paragliola r, pelizzo mr, testini m, de toma g. diagnostic, therapeutic and healthcare management protocols in parathyroid surgery: ii consensus conference of the italian association of endocrine surgery units. j endocrinol invest 2014;37(2):149-65. 3. miccoli p, materazzi g, baggiani a, miccoli m. mini-invasive video-assisted surgery of the thyroid and parathyroid glands: a 2011 update. j endocrinol invest. 2011;34(6):47380. 4. olson m.o. and dundr, m. nucleolus: structure and function. 2015. els. 1-9. 5. storck s, shukla m, dimitrov s, bouvet p. functions of the histone chaperone nucleolin in diseases. subcell biochem 2007;41:125-44. 6. box jk, paquet n, adams mn, boucher d, bolderson e, o'byrne kj, richard dj. nucleophosmin: from structure and function to disease development. bmc mol biol 2016;17(1):19. 7. ginisty h, sicard h, roger b, bouvet p. structure and functions of nucleolin. j cell sci 1999;112:761-7. 8. lieu d. cytopathologist-performed ultrasound guided fine-needle aspiration of parathyroid lesions. diagn cytopathol 2010;38:327-32. 9. tomić brzac h, bence-žigman z, dodig d. ultrazvuk vratnih organa. iv. poslijediplomski tečaj trajnog usavršavanja liječnika i kategorije. zagreb: medicinski fakultet sveučilišta u zagrebu, klinički zavod seemedj 2017, vol 1, no. 2 agnor counts in differential diagnosis of parathyroid adenoma and hyperplasia… 25 southeastern european medical journal, 2017; 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(in croatian) 14. fustar-preradovic l, sarcevic b, danic d. morphometry in differential diagnosis of pathologically altered parathyroid glands: adenoma and hyperplasia. coll antropol 2012;36(suppl 2):47-51. 15. tajrishi mm, tuteja r, tuteja n. nucleolin.the most abundant multifunctional phosphoprotein of nucleolus. commun integr biol 2011;4(3):267-75. 16. gaume x, place c, delage h, mongelard f, monier k, bouvet p. expression of nucleolin affects microtubule dynamics. plos one 2016;16;11(6):e0157534. 17. ploton d. structure and molecular organization of the nucleolus. zentralbl pathol 1994;140: 3-6. 18. egan mj, crocker j. nucleolar organiser regions in pathology. br j cancer 1992;65:17. 19. aubele m, biesterfeld s, derenzini m, hufnagl p, martin h, ofner d, ploton d, rüschoff j. guidelines of agnor quantitation. committee on agnor quantitation within european society of pathology. zentralbl pathol 1994;140:107-8. 20. hufnagl p, guski h, schulz hj. measuring of agnors using image analysis. zentralbl pathol 1994;140:31-5. 21. löwhagen t, sprenger e. cytologic presentation of thyroid tumors in aspiration biopsy smear. a review of 60 cases. acta cytol 1974;18:192-7. 22. van diest pj, baak jpa. morphometry. in: bibbo m, ed. comprehensive cytopathology. philadelphia: wb saunders, 1991:946-64. 23. boquist ll. nucleolar organizer regions in normal, hyperplastic and neoplastic parathyroid glands. virchows arch a pathol anat histopathol 1990;417:237-41. 24. mourad wa, pugh wc, huh yo, keating mj. cell kinetic analysis of interleukin-2 receptor-tested chronic lymphocytic leukemia using the agnor silver stain. am j clin pathol 1994;101:300-4. 25. rüschoff j, plate kh, contractor h, kern s, zimmermann r, thomas c. evaluation of nucleolar organizer regions (nors) by automatic image analysis: a contribution to standardization. j pathol 1990;161:113-8. 26. weeks sc, beroukas d, jarvis lr, whitehead r. video image analysis of agnor distribution in the normal and adenomatous colorectum. j pathol 1992;166:139-45. 27. tsai th, chang tc, chiang cp. nuclear area measurements of parathyroid adenoma, parathyroid hyperplasia and thyroid follicular adenoma. a comparison. anal quant cytol histol 1997;19:45-8. 28. tuccari g, abbona gc, giuffré g, papotti m, gasparri g, barresi g, bussolati g. agnor quantity as a prognostic tool in hyperplastic and neoplastic parathyroid glands. virchows arch 2000;437:298-303. seemedj 2017, vol 1, no. 2 agnor counts in differential diagnosis of parathyroid adenoma and hyperplasia… 26 southeastern european medical journal, 2017; 1(2) 29. kanematsu e, matsui h, deguchi t, yamamoto o, korematsu m, kobayashi a, nezasa si, yamamoto n, takeuchi t, tanaka t, kawada y. significance of agnor counts for distinguishing carcinoma from adenoma and hyperplasia in parathyroid gland. hum pathol 1997;28:421-7. seemedj 2018, vol 2, no. 1 to biofilm or not to biofilm? 12 southeastern european medical journal, 2018; 2(1) to biofilm or not to biofilm? valentina živković1, tomislav kurevija1, ivana haršanji drenjančević1,2, maja bogdan1,3, maja tomić paradžik1,4 jasminka talapko1, domagoj drenjančević1,2 1 faculty of medicine, josip juraj strossmayer university of osijek, osijek, croatia 2 university hospital center osijek, osijek, croatia 3 institute of public health osijek-baranja county, osijek, croatia 4 institute of public health brod-posavina county, slavonski brod, croatia corresponding author: domagoj drenjančević, md, phd domagoj@mefos.hr received: september 29, 2017; revised version accepted: april 3, 2018; published: november 27, 2018 keywords: biofilm, p. aeruginosa, s. aureus abstract aim: the goal of this research is to examine the biofilm forming ability of staphylococcus aureus and pseudomonas aeruginosa clinical isolates in different in vitro conditions using meuller-hinton and luria-bertani broths. material and methods: 30 strains of pseudomonas aeruginosa and 30 strains of staphylococcus aureus obtained from clinical specimens were used. after preparing the suspensions of bacteria inoculated on broths, they were set on microtiter plates and the biofilm production was measured using the spectrophotometric reader on 550 nm. strains were classified into four categories: nonproducing, weak producers, moderate and strong producers, based on the comparison of optical density of samples and negative control. results: both tested species successfully formed a biofilm in both broths (p<0.01). p. aeruginosa strains had a higher percentage of strong producers in both in vitro conditions, in comparison with s. aureus strains (3.3% vs 50%). nevertheless, there is no statistically significant difference in biofilm formation between the strains, regardless the used broths, and there is no statistically significant difference between the biofilm forming ability of both species observed separately regarding in vitro conditions either. conclusion: both species have an ability to produce biofilm, which likely contributes to the pathogenicity and virulence of these bacteria and also leads to a better understanding of their in vivo characteristics to cause infections related to biofilm. (zivkovic v, kurevija t, harsanji drenjancevic i, bogdan m, tomic paradzik m, talapko j, drenjančević, d. to biofilm or not to biofilm? seemedj 2018;2(1);12-19) mailto:domagoj@mefos.hr seemedj 2018, vol 2, no. 1 to biofilm or not to biofilm? 13 southeastern european medical journal, 2018; 2(1) introduction biofilm formation is one of the additional bacteria virulence factors which is still an interesting subject for numerous researches. biofilm infections are becoming a major health problem in chronic infections and implants. biofilm is a multicellular structure that protects bacteria from adverse environmental factors, making them highly resistant to different antibiotics. it also stores nutrients, which serve the bacteria to survive, protects them from phagocytosis, and secures survival in the host organism. resistance to disinfectants is a very important characteristic of biofilm because it prevents removing bacteria from the surface, enabling such microorganisms to permanently colonize the human organism with pathological consequences. biofilm should be considered as a mobile functional community with the features of a complete microorganism because, among other things, they have homeostasis, circulatory system, genetic material exchange and metabolic activity, which ensure their further development (1). in addition, biofilm-protected bacteria are capable to disperse individual bacterial cells and decomposing parts of biofilm into the surrounding tissues and circulation system. but most importantly, on the surfaces of medical devices or in the human body, biofilm is made by microorganisms with the ability to produce an extracellular polymeric substance. these polymeric substances have an ability to incorporate a large amount of water into their structure and become highly hydrated (2). these solid-liquid barriers between the surface and the aqueous environment allow the community of biofilms optimal conditions for the growth and survival of microorganisms. also, biofilm is formed exclusively by the cells that produce polysaccharides in sufficient quantity (3). several environmental and genetic signals control each step of biofilm development and dispersal. accumulation of signal molecules in the environment allows each bacterial cell to estimate cell density or the total number of bacteria at that time – the quorum detection or quorum sensing phenomenon. colonization of medical devices is proportionally increased by surface irregularity and microorganisms bond more rapidly to hydrophobic surfaces such as plastic, rather than hydrophilic ones. (2,4). the appearance of biofilm on implants and various surgical implantable devices causes chronic infections, rejection of implants, ineffectiveness of the embedded device, organ damage, and sometimes even lethal outcome for the patient. the aim of this research is to examine the biofilm forming ability of staphylococcus aureus and pseudomonas aeruginosa clinical isolates in different in vitro conditions using mueller-hinton and luria-bertani broths. material and methods sample preparation this study included 60 bacterial strains, 30 pseudomonas aeruginosa and 30 staphylococcus aureus strains, obtained from different clinical specimens from 2007 to 2015 and isolated in microbiological laboratories at university hospital center osijek, croatia and in the general hospital slavonski brod, croatia. all bacterial strains are part of the collection of microbial strains kept at the department of microbiology and parasitology, faculty of medicine, university of osijek. microorganisms were identified according to standard microbiological methods and biochemical tests to the species level (5). after the bacteria had been grown on the blood agar plate during 1824 hours incubation, two to three individual colonies of bacterial cultures were taken and inoculated into vials with 3 ml of mueller-hinton (mh) (becton dickinson and co., cockeysville md, usa) and luria-bertani (lb) (difco r luriabertani broth, becton dickinson, usa) broth. the suspensions were incubated in the thermostat at 37°c for another 18-24 hours. after incubation, the tubes were well mixed (vortexed) and 20 μl from each suspension was transferred into new tubes with 2 ml mh and lb broth, which yielded suspensions of approximately 5x105 cfu/ml concentrations. after the preparation, suspensions were planted on flat bottom seemedj 2018, vol 2, no. 1 to biofilm or not to biofilm? 14 southeastern european medical journal, 2018; 2(1) polyester microtiter plates (copan, brescia, italy). wells with 100 μl of uninoculated mh and lb were used as the negative control and the remaining wells had 50 μl mh or lb broths which were planted with 50 μl of the prepared suspensions. the biofilm-producing strain acinetobacter baumannii atcc 19606 was used as a positive control. the microtiter plate was incubated in the thermostat for 18-24 hours at 37°c. after the incubation, the broth was shaken out and wells were washed three times with distilled water. at the end of the experiment, coloring with 0.1% crystal violet and solubilization with 95% ethanol was done (6). all measurements were done in triplicate. quantification of biofilm the final step was a spectrophotometric measurement of biofilm production on an enzyme immunoassays plate reader (biorad 93200 pr3100 tsc microplate reader) at 550 nm. the optical density (od) values were measured in every well of the plate and they represent biofilm production. the final results were reported as the optical density cut-off value (odc), which was calculated as average od for each sample made in triplicate increased by three standard deviations of negative controls. the results were classified into the following categories: non-producers, weak, moderate and strong biofilm producers (6,7) according to the criteria presented in table 1. table 1. the criteria for evaluating biofilm production od = average optical density value of biofilm production in a single well; odc = limit value of biofilm production (at least some biofilm produced) statistical analysis the results were processed using the statistical software package spss 19.0 (ibm corp., armonk, ny, usa), and the data processing was carried out by checking normality distribution and calculation of descriptive data, including the frequencies, percentages, median and interquartile ranges. wilcoxon test of equivalent pairs, χ2 test with fisher's exact test and cramer's v (φ) coefficient were utilized for the statistical significance testing of the differences between two or more independent groups. results the biofilm production ability data for both bacterial species regarding the in vitro nutrient condition (incubation in mueller-hinton and luria-bertani broths) are shown in table 2. data are presented as the average of triplicate measurement of optical density and includes medians and interquartile ranges for each variable used. 𝑶𝑫 < 𝑶𝑫𝒄 non-producers 𝑶𝑫𝒄 < 𝑶𝑫 < 𝟐 × 𝑶𝑫𝒄 weak producers 𝟐 × 𝑶𝑫𝒄 < 𝑶𝑫 < 𝟒 × 𝑶𝑫𝒄 moderate producers 𝟒 × 𝑶𝑫𝒄 < 𝑶𝑫 strong producers seemedj 2018, vol 2, no. 1 to biofilm or not to biofilm? 15 southeastern european medical journal, 2018; 2(1) table 2. the amount of biofilm formed, presented as the average optical density for s. aureus and p. aeruginosa using luria-bertani and mueller-hinton broths, in comparison to control staphylococcus aureus c q luria-bertani broth control 0.059 0.014 od (ar) 0.085 0.031 mueller-hinton broth control 0.076 0.011 od (ar) 0.097 0.043 pseudomonas aeruginosa luria-bertani broth control 0.032 0.017 od (ar) 0.318 0.481 mueller-hinton broth control 0.070 0.07 od (ar) 0.330 0.602 legend: c = median; q = interquartile range; od (ar) = average optical density by comparing the data for both bacteria and cultivation media (table 2), it can be seen that interquartile dispersal is greater for p. aeruginosa than for s. aureus strains. table 3. distribution of biofilm production in s. aureus and p. aeruginosa strains using luria-bertani and mueller-hinton broth (p<0.01, wilcoxon's equivalent pair test) staphylococcus aureus non-producers weak producers moderate producers strong producers f (%) f (%) f (%) f (%) lb 17 (56.7) 13 (43.3) 0 (0) 0 (0) mh 19 (63.4) 9 (30) 1 (3.3) 1 (3.3) pseudomonas aeruginosa non-producers weak producers moderate producers strong producers f (%) f (%) f (%) f (%) lb 0 (0) 8 (26.7) 4 (13.3) 18 (60) mh 4 (13.3) 6 (20) 5 (16.7) 15 (50) legend: lb = luria-bertani broth; mh = mueller-hinton broth; f = frequency it has been found that both bacterial species, s. aureus and p. aeruginosa successfully (to a statistically significant degree) created biofilm in both cultivation media (p<0.01, wilcoxon's equivalent pair test). seemedj 2018, vol 2, no. 1 to biofilm or not to biofilm? 16 southeastern european medical journal, 2018; 2(1) table 4. the contingency table for biofilm production of p. aeruginosa and s. aureus strains in luriabertani and mueller-hinton broth. pseudomonas aeruginosa luria-bertani broth staphylococcus aureus weak producers moderate producers strong producers total non-producers 2 2 13 17 6.7% 6.7% 43.3% 56.7% weak producers 6 2 5 13 20.0% 6.7% 16.7% 43.3% total 8 4 18 30 26.7% 13.3% 60.0% 100.0% pseudomonas aeruginosa mueller-hinton broth staphylococcus aureus nonproducers weak producers moderate producers strong producers total nonproducers 3 3 3 10 19 10% 10.0% 10.0% 33.3% 63.3% weak producers 0 2 2 5 9 0% 6.7% 6.7% 16.7% 30.0% moderate producers 0 0 0 0 0 0% 0% 0% 0% 0% strong producers 0 1 0 0 1 0% 3.3% 0% 0% 3.3% total 4 6 5 15 30 13.3% 20.0% 16.7% 50.0% 100.0% the correlation between the tested bacterial species according to their biofilm production ability is shown in table 4. there is no statistically significant difference in biofilm formation between s. aureus and p. aeruginosa strains in luria-bertani (fischer's exact test, p=0.075) or in mueller-hinton broth (fischer's exact test, p=0.359). the ability to produce biofilm depending on different cultivation conditions is shown in figures 1 and 2. staphylococcus aureus strains had very modest biofilm production in both broths: 43.3% of the strains seem to be weak producers and the remaining are non-producers in luria-bertani broth. there is even a smaller number of biofilm weak producers (30%) in mueller-hinton broth, and almost all remaining ones are biofilm non-producers, with the exception of one moderate (3.3%) and one strong (3.3%) producer. p. aeruginosa strains belong to strong biofilm producers in both in vitro conditions. in luria-bertani broth, all tested strains were shown as biofilm producers. weak producers accounted for 26.7% of the strains, moderate ones accounted for 13.3%, and 60.0% were strong producers. there was 20.0% of weak producers, 16.7% of moderate producers and 50.0% of strong producers in mueller-hinton broth, and 13.3% of the strains were biofilm nonproducers. there was no statistically significant difference between cultivation conditions and the ability to form biofilm either in s. aureus or in p. aeruginosa strains. seemedj 2018, vol 2, no. 1 to biofilm or not to biofilm? 17 southeastern european medical journal, 2018; 2(1) figure 1. biofilm production ability of s. aureus in luria-bertani and mueller-hinton broths (fischer's exact test, p=0.664). figure 2. biofilm production ability of p. aeruginosa in luria-bertani and mueller-hinton broths (fischer's exact test, p=0.476). discussion the main observation which arises from this study is that both bacterial species, grampositive s. aureus and gram-negative p. aeruginosa, successfully and to a statistically significant degree form biofilm in both tested broths (table 4). another important observation is that interquartile dispersal is greater in p. aeruginosa compared to s. aureus strains. p. aeruginosa strains have a higher incidence of extreme values and thus a greater range of results. however, no statistical difference was observed with regard to the medium in which biofilm production was measured. both species showed that biofilm production is more pronounced in luria-bertani medium by comparing the percentage, but no statistical significance has been established in statistical tests. although luria-bertani medium is a medium in which higher production is expected, other authors have also pointed out the possibility that biofilm production may be unexpectedly expressed depending on the conditions of bacterial growth. biofilm formation can be strongly affected both by growth media and by temperature (8,9). another study (10) has also shown that both of these bacterial species are biofilm producers, independently of the clinical specimen isolation origin (sputum, urine, urine catheter, etc.). in this study, which involved the application of congo agar and tube method, 0 5 10 15 20 non-producers weak moderate strong s. aureus luria bertani mueller hinton 0 5 10 15 20 non-producers weak moderate strong p. aeruginosa luria bertani mueller hinton seemedj 2018, vol 2, no. 1 to biofilm or not to biofilm? 18 southeastern european medical journal, 2018; 2(1) influence of the different in vitro conditions on biofilm forming ability of these two bacterial species was visible. (10) both of the bacterial species have been shown to be strong producers of biofilm, with more than 80% of strong producers found (10). in our study, p. aeruginosa strains were strong producers in 55% cases, equally in both broths, whereas s. aureus strains had only one strong producer (3.3%). by comparing the results of this small series of experiments, it is reasonable to assume that the biofilm forming ability is greatly influenced by cultivation conditions, that it is nutrient dependent and also has a significant role in antimicrobial susceptibility of biofilms. (11,12,13) also, it is very important to emphasize that the role of biofilm in the genesis of infections associated with medical devices is indisputable. microorganisms isolated from the samples of patients with these infections often exhibit the apparent ability to generate biofilms, as has been shown in many studies. (14,15) additionally, it is known that multiple bacterial species can cooperate and form complex networks with many defending mechanisms and built-in sophisticated protection against the human immune system and antimicrobials as well. (16) such polymicrobial biofilms are nowadays recognized as a significant factor in the pathogenesis of multiple infections in humans. conclusion the obtained results are in agreement with previous medical and microbiological knowledge of biofilm formation, which plays a pivotal role in numerous infections such as periodontitis, chronic prostatitis, bacterial vaginosis, chronic otitis media, osteomyelitis, chronic pulmonary infections in cystic fibrosis patients, and chronic wound infection, considering that the investigated bacterial species, s. aureus and p. aeruginosa, are the most common etiological pathogens of these infections. funding we greatly appreciate the financial support of university of osijek, faculty of medicine (research grant vif2016-mefos-27). transparency declaration competing interests: none to declare. references 1. milanov d, ašanin r, vidić b, krnjajić d, petrović j. biofilm – organizacija života bakterija u prirodnim ekosistemima. arhiv veterinarske medicine 2008;1:5-15. 2. rodney md. biofilms: microbial life on surfaces. journal list. 2002;8:881–90. 3. irie y, roberts ael, kragh kn, gordon vd, hutchison j, allen rj et al. the pseudomonas aeruginosa psl polysaccharide is a social but noncheatable trait in biofilms. mbio 2017;8:00374-17. 4. characklis wg, mcfeters ga, marshall kc. physiological ecology in biofilm systems. new york: john wiley & sons; 1990. p. 341–94. 5. jorgensen jh, pfaller ma, carroll kc, funke g, landry ml, richter ss, warnock dw. manual of clinical microbiology. washington, dc, usa: asm press, 2015. 6. milanov d, bugarski d, petrović j, rackov o. primena testa na mikrotitracionim pločama i mikroskopskih tehnika u ispitivanju sposobnosti nekih bakterijskih vrsta izolovanih od životinja da formiraju biofilm. arhiv veterinarske medicine 2010;3:23-37. 7. ghellai l, hassaine h, klouche n, khadir a, aissaoui n, nas f et al. detection of biofilm formation of a collection of fifty strains of staphylococcus aureus isolated in algeria at the university hospital of tlemcen. j bacteriol res 2014;6:1-6. 8. labrie j, pelletier-jacques g, deslandes v, ramjeet m, auger e, nash jh, et al. effects of growth conditions on biofilm formation by actinobacillus pleuropneumonia. vet res 2010;41:3. 9. nucleo e, steffanoni l, fugazza g, migliavacca r, giacobone e, navarra a et al. growth in glucose-based medium and exposure to subinhibitory concentrations of imipenem induce biofilm formation in a multidrug-resistant clinical isolate of seemedj 2018, vol 2, no. 1 to biofilm or not to biofilm? 19 southeastern european medical journal, 2018; 2(1) acinetobacter baumannii. bmc microbiol 2009;9:270. 10. rewatkar ar, wadher bj. staphylococcus aureus and pseudomonas aeruginosa biofilm formation methods. iosr-jpbs 2013;8:36-40. 11. rochex a, lebeault jm. effects of nutrients on biofilm formation and detachment of a pseudomonas putida strain isolated from a paper machine. water res 2007;41:2885-92. 12. henry-stanley mj, hess dj, wells cl. aminoglycoside inhibition of staphylococcus aureus biofilm formation is nutrient dependent. j med microbiol 2014;63:861-9. 13. bogdan m, drenjancevic d, harsanji drenjancevic i, bedenic b, zujic atalic v, talapko j et al. in vitro effect of subminimal inhibitory concentrations of antibiotics on the biofilm formation ability of acinetobacter baumannii clinical isolates. j chemother 2018;30:16-24. 14. pour nk, dusane dh, dhakephalkar pk, zamin fr, zinjarde ss, chopade ba. biofilm formation by acinetobacter baumannii strains isolated from urinary tract infection and urinary catheters. fems immunol med microbiol 2011;62:328-38. 15. rodríguez-baño j, martí s, soto s, fernándezcuenca f, cisneros jm, pachón j, et al. biofilm formation in acinetobacter baumannii: associated features and clinical implications. clin microbiol infect. 2008;14:276–8. 16. hotterbeekx a, kumar-singh s, goossens h, malhotra-kumar s. in vivo and in vitro interactions between pseudomonas aeruginosa and staphylococcus spp. front cell infect microbiol 2017;7:106. doi: 10.3389/fcimb.2017.00106. ecollection 2017. seemedj 2019, vol 3, no. 1 employment in patients with renal replacement therapy 11 southeastern european medical journal, 2019; 3(1) original article employment in patients with renal replacement therapy 1 mirjana đukić 1, 2, lada zibar 2, 3 1 general county hospital vinkovci, vinkovci, croatia 2 faculty of medicine, josip juraj strossmayer university of osijek, osijek, croatia 3 department of nephrology, university hospital centre osijek, osijek, croatia corresponding author: lada zibar, ladazibar@gmail.com received: january 14, 2018; revised version accepted: april 29, 2019; published: may 31, 2019 keywords: employment; chronic kidney disease; kidney transplantation; renal replacement therapy; work capacity abstract aim: to determine the prevalence and rate of employment of patients on renal replacement therapy (rrt) for end-stage renal disease (esrd), to study the difference in the rate of employment between patients on hemodialysis (hd) and those with functioning kidney transplant (fkt) and to compare the rate of employment with patients’ opinions about their working ability and determine the possible reasons for the presumed disproportion.. methods: 220 rrt patients (126 on hd and 94 with fkt) at the university hospital centre osijek were surveyed. we created and used a questionnaire about the level of education, occupation, employment, professional timeline during the course of rrt, personal opinion about working ability and potential reasons for unemployment. research was conducted during april and may 2017. the data were analyzed using spss (version 16.0. inc., chicago, il, usa). results: at the time of our research, 13.7% of patients on rrt were employed. employment of fkt patients prevailed, without significant difference compared with dialyzed patients of working age (15 to 65 years old). 38.3% of patients in that age group felt capable of working. transplantation did not improve access to employment. highly educated people were employed more frequently. the main reasons for unemployment were poor health caused by ckd, advanced age, and employers’ unwillingness to hire chronically ill persons because of the potential need to adjust working hours. conclusion: ckd reduced working ability and employment opportunities. only a minority of patients on rrt were employed. kidney transplantation did not increase the rate of employment. patients should therefore be provided with education, appropriate guidelines and support for finding employment. (đukić m, zibar l. employment in patients with renal replacement therapy. seemedj 2019; 3(1); 1120) seemedj 2019, vol 3, no. 1 employment in patients with renal replacement therapy 12 southeastern european medical journal, 2019; 3(1) introduction chronic kidney disease (ckd) often develops slowly and without specific symptoms. consequently, many patients are diagnosed in the advanced stages of the disease (1). ckd is accompanied by numerous complications: anemia, mineral and bone disorders, inflammation-induced progression of atherosclerosis, malnutrition, dyslipidemia, cardiovascular disease (cvd), infections, immune system disorders, gastrointestinal disorders, neurological disorders, etc. (2). patients with end-stage renal disease (esrd) are treated by renal replacement therapy (rrt) (1). rrt is performed by dialysis or renal transplantation (tx). today, more than a million patients with ckd worldwide are treated by hemodialysis (hd). during and after the hd procedure, numerous complications such as hypotension, cramps, nausea, vomiting, headache, tingling, sweating or hypoglycemia may occur (3). hypotension is one of the most common complications (4). tx is the best protocol for treating esrd (5). successful kidney tx improves quality of life (qol) and reduces the risk of death for most patients, when compared with the risk present for patients on dialysis (6, 7, 8). the term “health-associated qol” has been in use (hrqol, health-related quality of life) for several decades. this term encompasses the effect of health, i.e., of the illness and of treatment for the illness on the patient’s physical, cognitive and social functioning. ckd significantly affects qol. hd is a complex procedure that requires frequent visits to the dialysis center, usually 3 times per week, which takes time and necessitates changes in the lifestyle. therefore, hd patients have impaired qol, both from symptoms of esrd and from the psychological and physical burdens of hd. patients who develop esrd are in poor physical condition, they develop anemia, get tired quickly, often have sleep disorders, and develop depression (9, 10). although qol cannot be measured directly, qol questionnaires are designed according to determinants that are important to the patients. qol questionnaires survey the physical and mental condition and the characteristics of the primary renal disease which affect qol. studies using such or similar questionnaires have shown that qol of hd patients is significantly worse than that of the general population, especially in regard to the physical component of hrqol (9). tx, the best form of rrt, improves qol, but it could be hard to find a suitable graft and to have the graft successfully accepted (11, 12). likewise, lifelong immunosuppressive therapy after tx carries risks of infection, malignancy, and cvd. working ability is considered a significant indicator of well-being and health (13). although patients have significantly improved health after tx, in practice, transplanted patients frequently face unemployment. we thus presumed that treatment of ckd with dialysis or tx prevents patients from gaining employment and that kidney tx, as the best form of rrt, does not improve employment access for patients. the aims of the study were to determine the prevalence of employment of patients on and during the course of rrt, to evaluate whether there was a difference between the prevalence of employment for patients on hd and those after tx, to compare employment with patients’ opinions about their working ability, and to establish the possible reasons for the presumed disproportion. patients and methods the research was conducted at the department of nephrology of the clinic for internal medicine at the university hospital centre osijek during april and may 2017, with approval of the ethics committee for research at the faculty of medicine, josip juraj strossmayer university in osijek, croatia. 220 patients were selected as the subjects of our study: 126 were treated by hd and 94 by functioning kidney transplant (fkt). at the time of our research, the sample included cooperative patients that could and did fill in the questionnaire (the initial eligible group consisted of 150 hd and 200 fkt patients). out of 126 patients who were treated by hd during the research period, 12 had undergone a kidney transplant. 133 (60.5%) of all participants were male and 87 (39.5%) were female. the median age was 63, ranging from 31 to 88 (iqr 52 − 71). 80 (63.5%) of the 126 participants treated by seemedj 2019, vol 3, no. 1 employment in patients with renal replacement therapy 13 southeastern european medical journal, 2019; 3(1) dialysis were male and 46 (46.5%) were female. the median age of patients on hd was 69, ranging from 32 to 88 (iqr 59 − 76). of 94 patients with fkt, 53 (56.4%) were male and 41 (43.6%) female. the median age of patients with fkt was 57, ranging from 31 to 78 (iqr 50 − 63). 120 patients were of working age (15 − 65 years old). chronic glomerulonephritis and diabetic nephropathy were the two most common causes of renal failure in patients on rrt. the third most common basic renal disease was arterial hypertension, followed by interstitial nephritis and autosomal dominant polycystic kidney disease. patients were asked to fill in a questionnaire about their formal education, occupation, employment, professional timeline during the course of rrt and possible reasons for unemployment. the questionnaire was specifically created for our study by the researcher and it included data on finding employment and carrying out work along the following timeline: before ckd diagnosis, after ckd diagnosis, prior to starting dialysis, a year subsequent to starting dialysis, before tx, a year after tx and at the time of the research (figure 1). figure 1. time points for which the participants were asked about their employment demographic data, time data about the dialysis and tx, as well as data about the primary renal disease and concomitant morbidity were taken from medical records. data were statistically analyzed using spss (version 16.0. spss inc., chicago, il, usa). descriptive statistics included the median with interquartile range (iqr) for numeric data. absolute and relative frequencies were used for nominal data. differences between two unrelated groups were obtained by the chi-square or mann-whitney test, depending on the data type. marginal homogeneity was used for the difference between two related samples. statistical significance was accepted if p was < 0.05. results 220 patients were included in the research, 126 of which were treated with hd and 94 with fkt. fkt patients were significantly younger than patients on dialysis at the time of the research (p < 0.01; mann-whitney test). diabetic nephropathy (27%) and arterial hypertension (23%) were two of the most common causes of renal failure in patients on hd. chronic glomerulonephritis (43.6%) and autosomal dominant polycystic kidney disease (19.1%) were two of the most common causes of renal failure in patients with fkt (figure 2). seemedj 2019, vol 3, no. 1 employment in patients with renal replacement therapy 14 southeastern european medical journal, 2019; 3(1) *chi-square test figure 2. causes of renal failure in patients on hemodialysis (hd), n = 126, and with functioning kidney transplant (fkt), n = 94 of the 220 participants, 71 completed only primary education or less (32.3%), 118 finished high school (53.6%), and 31 had a higher education qualification or a university degree (14.1%). after receiving the diagnosis of ckd, employment among the working patients decreased by 32.7% (103 were working before the diagnosis, 70 after). overall employment decreased by 4.7% (47.4% before the diagnosis, 42.7% thereafter). the number of subjects on sick leave (for more than a month) increased 24 times (1 before the diagnosis, 24 immediately thereafter) and 4 people started receiving disability benefits because of ckd immediately after the diagnosis (figure 3). *the marginal homogeneity test figure 3. employment before and after diagnosis of chronic kidney disease (ckd) 0 5 10 15 20 25 30 35 40 45 diabetic nephropathy arterial hypertension chronic glomerulonephritis interstitial nephritis autosomal dominant polycystic kidney disease n hd tx *p < 0.001 0 20 40 60 80 100 120 working full time working part time sick leave unemployed disability benefits n before diagnosis of ckd after diagnosis of ckd seemedj 2019, vol 3, no. 1 employment in patients with renal replacement therapy 15 southeastern european medical journal, 2019; 3(1) immediately after the diagnosis of ckd and before the beginning of hd, the number of working patients decreased by 22.9%, and overall employment decreased by 9.6% (42.7% after the diagnosis of ckd, 33.1% before hd). the number of recipients of disability benefits increased by 4.5% (4 after the diagnosis of ckd, 18 before the start of hd). the proportion of employees decreased by 50% after the start of hd treatment when compared to the period before hd (54 employed before hd, 27 a year after the start of hd). overall employment decreased by 8.1% (33.1% were employed before hd, 25% a year after starting hd). out of all employed patients, 50.1% were on sick leave for more than a month after the beginning of hd. the number of recipients of disability benefits increased by 7.7% compared to the period before dialysis (figure 4). *the marginal homogeneity test figure 4. employment before starting dialysis treatment and within the first year of dialysis the number of patients who were working a year after tx increased by 9.1% compared to the period before tx (20 before tx, 22 after tx). overall employment increased by 0.9% (27.3% were employed before tx, 28.2% a year after tx; figure 5). *the marginal homogeneity test figure 5. employment before and a year after kidney transplantation (tx, n = 94) 0 5 10 15 20 25 30 35 40 working full time working part time sick leave unemployed disability benefits n before tx after tx *p = 0.916 0 10 20 30 40 50 60 working full time working part time sick leave unemployed disability benefits n before hd first year of hd *p = 0.001 seemedj 2019, vol 3, no. 1 employment in patients with renal replacement therapy 16 southeastern european medical journal, 2019; 3(1) at the time of the research, the number of employed participants had decreased by 36.4% compared to the time point a year after tx (22 after tx, 14 at the time of the research; figure 6). *the marginal homogeneity test figure 6. employment a year after transplantation (tx) and at the time of research (n = 94) overall employment decreased by 13.8% (31.9% after tx, 18.1% at the time of the research). the number of subjects using sick leave decreased by 5.3% in the same period and the number of disability benefits increased by 3.2%. at the time of the research, overall employment of all patients had decreased by 33.7% compared to the period before the diagnosis of ckd (47.4% of the participants had been employed before the diagnosis, 13.7% were employed at the time of the research). out of all patients who were employed at the time of the research, 31% had been on sick leave for more than a month (figure 7). *the marginal homogeneity test figure 7. employment before diagnosis of chronic kidney disease (ckd) and at the time of the research (n = 220) 0 5 10 15 20 25 30 35 40 45 working full time working part time sick leave unemployed disability benefitsn after tx at the time of research 0 20 40 60 80 100 120 working full time working part time sick leave unemployed disability benefitsn before diagnosis of ckd today *p < 0.001 seemedj 2019, vol 3, no. 1 employment in patients with renal replacement therapy 17 southeastern european medical journal, 2019; 3(1) the number of working patients on dialysis at the time of the research decreased by 82.5% compared to the period before ckd diagnosis and overall employment decreased by 21.3% (31.7% before diagnosis, 10.4% at the time of the research). out of all patients on hd who were employed at the time of the research, 6 were on sick leave for more than a month (46.2%). ten participants (7.9%) were receiving disability benefits because of ckd, 94 (74.6%) were retired due to age, disability caused by another illness or were military retirees. the number of working patients with fkt at the time of the research decreased by 77.8% compared to the period before the diagnosis of ckd and overall employment decreased by 50.1% (68.2% before the diagnosis, 18.1% at the time of the research). out of all fkt patients who were employed at the time of the research, 3 were on sick leave for more than a month (17.6%). 39 (41.4%) were receiving disability benefits, 23 (24.5%) were retired due to age, disability caused by another illness or were military retirees. of all participants of working age (15–65 ), 30 were employed (25%) while 46 felt capable of working (38.3%) at the time of the research (figure 8). figure 8. employment and feeling capable of working in patients of working age (15 to 65, n = 120) thirty percent of all employees in that age group were on sick leave for more than a month at the time of the research. 17 fkt patients aged 15 to 65 (22.3%) were employed, while 32 of them (47.9%) felt capable of working. out of the 17 patients employed at the time, 3 had been on sick leave for more than a month. 12 patients on hd aged 15 to 65 (26.1%) were employed at the time while 14 felt capable of working (30.4%). out of 12 employed patients, 6 had been on sick leave for more than a month. almost a quarter (24.2%, n = 22) of the unemployed patients aged 15 to 65 on rrt felt capable of working at the time of the research. 18.2% of those patients were retired, 4.6% were housewives, 36.4% were unemployed and 40.9% were receiving disability benefits because of ckd. as the main reason for their unemployment, the majority of respondents, 45.8%, reported poor health due to ckd, followed by advanced age (44.2%). the third main reason for unemployment was that employers did not want to hire someone with ckd because of the potential need to adjust working hours (5.8%). only 2.8% of patients with completed primary education were employed at the time of the research, in comparison with 14.4% of those with high school education and 35.5% of those with a university degree. discussion employment of patients on rrt worsened with the progression of ckd. overall employment decreased by 4.7% after diagnosis of ckd, and the number of persons on sick leave increased from 1 to 24. four people started receiving disability benefits immediately after the diagnosis. hd takes time, leads to a certain degree of disability, and often threatens 0 5 10 15 20 25 30 35 40 45 50 n employed feeling capable of working *p = 0.016 seemedj 2019, vol 3, no. 1 employment in patients with renal replacement therapy 18 southeastern european medical journal, 2019; 3(1) employment within a particular profession. accordingly, as patients started with hd, the number of employed patients decreased. within the first year of hd treatment, overall employment decreased by 8.1% when compared with the period before hd. 25% of patients were employed, and 50.1% of patients were on sick leave for more than a month after starting hd. the number of recipients of disability benefits continued to increase. within the first year after tx, the rate of employment of patients did not change significantly. if we compare the prevalence of employment after tx and at the time of the research, we see that overall employment decreased by 13.8%, although it would be logical to expect an increase in the number of people employed after tx, especially a year after tx, when patients have recovered from the kidney transplant procedure, since tx improves qol and working ability (14). patients with fkt have better health than those on hd, but overall employment of patients with fkt exhibited a twofold decrease in comparison with the period when they were on dialysis, during the rrt timeline from diagnosis of the disease to the present. despite that, at the time of the research, the cross-sectional prevalence of employment for fkt patients (18.1%) was higher than for those on hd (10.4%). this can be explained by the fact that current fkt patients were younger and more of them were employed at the beginning of ckd. this was also confirmed by the prevalence of employment of fkt and dialyzed patients of working age, 15 to 65. prevalence of employment was almost the same in both groups, but there was significant disproportion in the personal opinion about working ability between fkt and dialyzed patients: 30.4% of patients on hd and 47.9% of fkt patients felt capable of working. the number of fkt patients receiving disability benefits because of ckd was five times greater than that of dialyzed patients at the time of the research. almost three quarters (74.6%) of dialyzed patients had retired due to age, disability caused by another illness, or were military retirees. the ratio of disability and age-related retirement between fkt and dialyzed patients was in accordance with the age differences between those groups. a large number of disability benefits can be the result of availability of disability benefits and lack of support for patients on rrt who are willing to work. today, 13.7% of rrt patients are employed, which is a third (33.7%) less than was the case before they received the diagnosis of ckd. out of the total number of unemployed patients, almost a quarter felt capable of working. at the time of the research, a quarter of all patients aged 15 to 65 were employed, and almost 40% of the patients felt capable of working. 30% of the total number of employees in the group were on sick leave for more than a month, which means that the number of people working at that time was even smaller. we wondered what caused the disproportion between employment and feeling capable of working. as the main reason for unemployment, the majority of respondents reported poor health due to ckd, followed by advancing age and the fact that employers did not want to hire someone with ckd because of the potential need to adjust working hours. higher level of education had a favorable impact on the employment of our respondents. a similar research had been conducted in the rijeka university hospital, where the working ability of patients on hd was surveyed. that research showed that 22.4% of the patients had been employed at the beginning of hd. during the first year of hd, 36.1% of patients who had jobs at the beginning of dialysis were forced to retire, and there was also a disproportion between the number of employed patients and those who felt capable of working (15). based on our research and on the research conducted in rijeka, we can conclude that the number of working patients in the republic of croatia significantly decreases with the start of hd treatment. there is a large number of publications devoted to this topic – qol, working ability and possibility of employment of patients with ckd (14, 16, 17, 18). a study from the netherlands showed that tx significantly increased the employment rate of patients, which reverted to almost the same rate as prior to diagnosis of ckd. at the time of the study, 67% of fkt patients aged 15 to 65 were employed, as opposed to the situation in osijek, where only 22.3% of fkt patients from the same age group seemedj 2019, vol 3, no. 1 employment in patients with renal replacement therapy 19 southeastern european medical journal, 2019; 3(1) were employed. it is important to note that 45% of patients employed after tx in the netherlands had part-time jobs, while none of the transplanted respondents in osijek had a parttime job (14). the question is: are patients in croatia given the opportunity to work after tx or is retirement the only option? nowadays, the goal of medical treatment is not just survival, but also improvement in qol. important determinants of qol are the ability to work and access to employment. ckd disrupts qol, but patients who accept rrt do not give up on life; in fact, they do just the opposite – they choose to live. many patients with esrd, faced with limitations of health status and requirements of their treatment, reasonably decide to quit their jobs. this decision is made easier by the availability of disability benefits due to ckd. however, other patients who have the desire and opportunity to work find that guidance and support for employment are limited (19). these patients should be involved in public life and motivated to continue with their normal lives. a potential method for achieving higher employment of patients on rrt could be motivating the employers with financial incentives or other rewards. acknowledgement. none disclosure funding. no specific funding was received for this study. competing interests. none to declare references 1. vrhovac b, jakšić b, reiner, vucelić b. interna medicina. 4. izd. zagreb: medicinska biblioteka; 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7(1) review article computer vision solutions for range of motion assessment 1 jelena aleksic* university of belgrade, faculty of sport and physical education, the research centre, belgrade, serbia *corresponding author: jelena aleksic, jelena.aleksic@fsfv.bg.ac.rs received: mar 29, 2023; revised version accepted: apr 17, 2023; published: apr 30, 2023 keywords: joint range of motion, rehabilitation, computer vision system, machine learning, motion capture abstract joint range of motion (rom) is an important indicator of physical functionality and musculoskeletal health. in sports, athletes require adequate levels of joint mobility to minimize the risk of injuries and maximize performance, while in rehabilitation, restoring joint rom is essential for faster recovery and improved physical function. traditional methods for measuring rom include goniometry, inclinometry and visual estimation; all of which are limited in accuracy due to the subjective nature of the assessment. with the rapid development of technology, new systems based on computer vision are continuously introduced as a possible solution for more objective and accurate measurements of the range of motion. therefore, this article aimed to evaluate novel computer vision-based systems based on their accuracy and practical applicability for a range of motion assessment. the review covers a variety of systems, including motion-capture systems (2d and 3d cameras), rgb-depth cameras, commercial software systems and smartphone apps. furthermore, this article also highlights the potential limitations of these systems and explores their potential future applications in sports and rehabilitation. (aleksic j*. computer vision solutions for range of motion assessment. seemedj 2023; 7(1); 55-66) seemedj 2023, vol 7, no 1 computer vision for range of motion 56 southeastern european medical journal, 2023; 7(1) introduction joint range of motion (rom) is an important indicator of physical functionality and musculoskeletal health (44). it plays a vital role in the sports field, as athletes require adequate levels of joint mobility to reduce the risk of injuries and maximize their performance (57, 12). similarly, restoring the joint range of motion in rehabilitation is essential for faster recovery and better physical functionality (16, 32, 68). therefore, assessment of the joint range of motion is necessary for determining the limitations in joint mobility and creating treatment or training plans that can effectively enhance joint functionality, reduce the risk of injury and maintain optimal levels of performance (40, 51). common traditional methods of measuring the range of motion in physical therapy include using goniometers and inclinometers, as well as visual estimation through various mobility tests (38, 52). the goniometer is widely considered the standard tool (i.e. the golden standard) for evaluating a range of motion in clinical settings (52). this tool measures the angle of joints by positioning the arms of the goniometer along the joint’s axis. to obtain a more comprehensive and accurate assessment of the joint range of motion, goniometers are often used in conjunction with inclinometers; instruments that measure the position of body segments (38, 52). finally, a method that is used when specialized tools such as goniometers and inclinometers are not available is a visual estimation of joint mobility during various flexibility tests. this method can provide a quick estimation of the joint range of motion; however, it greatly depends on the rater’s ability to visually determine the degree of motion (23). while all of these methods can provide some degree of accuracy, their estimation is subjective as it may vary depending on the observer’s perception and experience or placement of the tool (1). developments in technology have now opened the possibility of using computer vision for a more objective range of motion assessment in sports and rehabilitation. computer vision (cv) is a new technology that employs several techniques such as pattern recognition, machine learning and image processing, to extract meaningful information from visual data. this technology can automate many tasks that require subjective assessment, potentially making them more efficient and accurate (35). current computer vision-based systems have mainly been developed for entertainment purposes (i.e. the gaming industry), although there have been many successful attempts to apply these systems in sports and rehabilitation (48). a common issue with these systems is achieving accurate pose-estimation results, which has limited their use in a range of motion assessment (7). the rapid advancements of this technology continually introduce new possible solutions, highlighting the importance of constantly assessing the reliability and validity of these novel systems. therefore, this study aims to present an up-to-date review of currently available computer vision-based systems, focusing on their reliability and validity in range of motion assessment, as well as their practical applicability. computer vision-based range of motion solutions the use of computer vision-based systems offers several advantages over the aforementioned traditional range of motion assessment methods: 1) computer vision-based systems eliminate the need for physical contact with the patient, making the assessment more comfortable and efficient (35); 2) these systems can capture a large amount of data in a short period, therefore providing detailed and objective quantitative measurements (56); 3) moreover, computer vision systems can track a range of motion both in real-time and over a longer period, which enables trainers and clinicians to monitor progress and adjust training or treatment plans accordingly (60). this paragraph will discuss some of the most commonly used computer vision systems for a seemedj 2023, vol 7, no 1 computer vision for range of motion 57 southeastern european medical journal, 2023; 7(1) range of motion assessment, including motion capture cameras (i.e. 2d, 3d and rgb-depth cameras), commercial software systems and smartphone apps (table 1). table 1. computer vision systems used for range of motion assessment technology device / app studies (authors) rom assessed reliability/ validity price markerbased 3d systems vicon; qualisys faber et al. (2009); inokuchi et al., 2015; me et al., 1998 shoulder, neck, lower extremities icc= 0.78-0.98 r = 0.779–0.863 (compared to crom device) >$10,000 markerless 3d systems dari motion; the captury cabarkapa et al., 2022; fleisig et al., 2022; harsted et al., 2019 hip, knee and ankle; shoulder and elbow icc= 0.64-0.92 r=0.74-0.99 (compared to vicon) >$10,000 rgb-depth cameras kinect beshara et al., 2020, 2021; cai et al., 2019; hawi et al., 2014; mortazavi et al., 2018; özsoy et al., 2022; zulkarnain et al., 2017 shoulder and lower extremities icc=0.62-0.98 r= 0.73-0.97 (compared to vicon) ~$399 commercial software systems kinetisense macaulay, 2017 shoulder and hip; elbow/wrist knee/ankle icc= 0.85-0.96 icc= 0.61-0.69 >$1,000 smartphone apps goniometer pro pourahmadi et al., 2016; wellmon et al., 2016 wrist and shoulder icc= 0.79-0.82 r≥ 0.80 (compared to goniometer and inclinometer) ~$9.99 combination of systems mira software + kinect wilson et al., 2017 shoulder r= 0.96-0.99 (compared to vicon) >$1,000 seemedj 2023, vol 7, no 1 computer vision for range of motion 58 southeastern european medical journal, 2023; 7(1) motion-capture (mocap) camera systems motion capture (mocap) cameras are advanced systems that capture and record movement in two-dimensional or three-dimensional spaces. some of these systems require the placement of passive markers on the body which reflect infrared light emitted by the cameras (31). in contrast, other systems can automatically detect anatomical markers through depth-offield sensors or deep-learning-based algorithms (7). regardless of the need for active or passive marker placement, motion-capture systems have the potential to be used for a range of motion assessment as they can track the movement of anatomical segments of the body. however, the accuracy of measurements can vary depending on the motion-capture technology integrated with the system (7). based on previous research, some of the most common motion-capture systems that are used for a range of motion assessment include: 2d and 3d systems. these systems use highspeed cameras and specialized software to track the position and movement of the body in a two-dimensional or three-dimensional space (31). the main difference between these two systems is that 2d cameras can track objects and record movement along a two-dimensional axis (i.e. height and width), while 3d cameras also include a third dimension (i.e. depth of field) (63). to estimate the position of body segments and human motion, 2d systems use the techniques of direct regression to identify key points on the body or heat maps to represent the probability of a joint being located at a particular position (9, 70). however, it is important to acknowledge that 2d systems lack the precision and accuracy of 3d motion capture systems, as they do not include the dimension of depth, which is important for a more comprehensive range of motion assessment (i.e. measuring rotation) (15, 63). therefore, marker-based 3d motion capture systems are highly regarded as the preferred method for a range of motion assessment (6). these systems use marker-based or markerless pose estimation techniques to capture and analyze movement (4, 34). marker-based systems (i.e. vicon and qualisys) capture the position of markers that reflect the infrared light emitted from the cameras (30). when these markers are placed on specific anatomical landmarks on the body, this reveals the position and orientation of each marker in a three-dimensional space and allows the system to precisely determine the joint orientation (34). normally, time-of-flight (tof), triangulation techniques and machine-learning algorithms are used to calculate the position of each marker and estimate human motion with more precision and detail in real-time (31). a crucial step in this process is targeted marker placement around the joint segments of the body (34), which can greatly affect the accuracy of data obtained from the cameras. however, this is usually a time-consuming process that is not very practical for collecting data outside laboratory settings (4, 5). this poses a significant advantage of markerless systems (i.e. dari motion and captury) as they greatly reduce the time required to prepare the subject for testing and facilitate data collection on the field (4, 5). previous studies have demonstrated good to excellent reliability (icc>0.80) of dari motion (dari motion, overland park, ks, usa) (10) in measuring the range of motion related to squat exercise (4, 5). however, only one study directly compared the accuracy of this system to a marker-based system, specifically in relation to baseball pitching-related range of motion (14). the results of this study showed that while the internal consistency of joint angle measurements was good (icc=0.64–0.92), the magnitudes of angle measurements differed between systems for up to 16 degrees (14). another study compared the validity of the captury (the captury gmbh, saarbrüken, germany) (65) markerless system to vicon (i.e. the golden standard) and found strong correlations for all range of motion measurements related to squat (r=0.74–0.99) and jump exercise (r=0.63–0.98) (19). while markerless systems certainly show promising results and better practicality of use, marker-based systems are still widely considered the golden standard in human motion analysis (19). moreover, the cost of both seemedj 2023, vol 7, no 1 computer vision for range of motion 59 southeastern european medical journal, 2023; 7(1) marker-based and markerless systems (~$10,000–$300,000 price per unit) remains a significant obstacle to their broad implementation in sports and rehabilitation (63). rgb-depth camera systems are innovative, lowcost solutions that combine the technology of 3d motion analysis with the practicality of use. these systems integrate depth-of-field sensors, which can calculate the distance of each point from the camera and create a three-dimensional representation of the model (22). the techniques for determining the dimension of depth may vary between camera models. some systems use a narrow-baseline binocular stereo vision technique to estimate the depth dimension by gathering multiple 2d captures (i.e. pointgrey bumblebee and stereolabs zed camera) (22). while other, more advanced systems use timeof-flight (tof) and infrared (ir) technology to determine the depth of field by calculating the time needed for light to travel between two points (i.e. microsoft azure kinect) (7, 62). these systems also implement machine learning algorithms to estimate the position of joints and track human motion without the need for the placement of reflective markers (47). although systems such as kinect (microsoft corp., redmond, wa, usa) were originally designed for gaming and virtual reality purposes, researchers have identified their potential for implementation in sports and rehabilitation due to their markerless pose estimation technology (2). previous studies found moderate-to-good intraand inter-rater reliability (icc=0.62–0.99) for shoulder range of motion measurements (2, 3, 8, 20, 21, 36, 49, 55, 73). at the same time, validity varied from poor to excellent for active shoulder range of motion compared to a video motion capture system (r=0.53) (59), lateral photographs (r= 0.33–0.79) (42) and vicon 3d motion capture system (r=0.73–0.97) (6). similarly, good to excellent agreement was found between kinect and vicon for lower extremities flexion and extension measurements (24, 33), while poor agreement was found for rotational movements (33). these results certainly highlight the vast potential of rgb-depth cameras being used for a range of motion assessment in the future; moreover, considering their portable, lightweight design and affordable price (~$399 per unit), these systems pose a much more accessible option for widespread use. however, considering the lack of studies and varied results, further research is needed to better assess their accuracy before fully adopting these systems in sports and rehabilitation. commercial software systems commercial software systems based on computer vision are becoming increasingly popular in sports and rehabilitation. these systems use advanced statistical algorithms and deep-learning frameworks, which are able to interpret and predict visual data, recognize anatomical segments and analyze human motion (50). one of the main advantages of these systems is that they can be paired with various types of video-capturing devices (i.e. standard video cameras or motion-capture cameras) to provide enhanced precision and more detailed measurements of human movement (41). some of these systems may also include their proprietary sensors or cameras (i.e. vicon nexus) developed to work seamlessly with the software (37). given the increasing number of commercial software systems available on the market, this review will specifically focus on software systems developed or researched for a range of motion assessment. these systems include: vicon nexus (vicon, oxford metrics ltd., oxford, uk) is the gold standard in advanced kinematic data analysis (37). this software processes signals from reflective markers captured with vicon 3d motion-capture cameras, allowing vicon nexus to create a 3d representation of the model, which can be viewed and analyzed from any angle (64). the software also provides various tools for measuring joint angles and calculating complex kinematic and kinetic data (i.e. force, velocity, acceleration and inverse kinematics). seemedj 2023, vol 7, no 1 computer vision for range of motion 60 southeastern european medical journal, 2023; 7(1) kinetisense (kinetisense inc.) is a software designed to upgrade the default algorithm included in the microsoft software developer kit by offering an advanced range of motion algorithm. this software can be paired with microsoft kinect systems for kinematic analysis (39). although there is a lack of studies assessing the accuracy of this software, one study showed good reliability of kinetisense in measuring shoulder and hip range of motion (icc=0.85– 0.96) and moderate reliability in measuring elbow/wrist and knee/ankle range of motion (icc=0.61–0.69) (39). theia3d (theia markerless inc.) is a commercial software package that offers a markerless motion-capture solution for a range of motion assessments, among many other types of activities. this software can be integrated with 2d cameras to capture movement and then biomechanically analyze human motion based on computer vision and machine-learning algorithms. this software calculates the 3d coordinates of each anatomical segment by estimating their 2d locations on each frame and then recreates a 3d model of the body (29). ipi mocap studio software (ipi soft llc) is a computer vision-based software that can be paired with 2d or rgb-depth cameras to extract spatiotemporal information, track movement and automatically pinpoint up to 16 anatomical markers at a time (33). previous studies found that ipi mocap studio software can be used as a valid tool for measuring the hip and knee range of motion in the sagittal and frontal planes (33). medical interactive recovery assistant (mira; mira rehab ltd., london, uk) is a software platform originally designed for exergaming and telerehabilitation purposes to help patients recover faster from injuries. this software integrates a range of motion measurement tools and requires depth-sensing cameras (i.e. kinect) to capture and analyze movement data in order to help therapists assess patients virtually (72). a study by wilson et al. (2017) evaluated the validity of mira software paired with a kinect camera for shoulder range of motion assessment. it showed very strong correlation results between measurements obtained from mira+kinect (r=0.96–0.99) and vicon 3d motion capture system (72). despite the numerous advantages of these systems, their cost (>$1,000–10,000) remains a significant obstacle against their potential widespread use for a range of motion assessment in sports and rehabilitation. while low-cost or free alternatives are available in the form of open-source software (i.e. openpose, opencam and free mocap) or smartphone apps, their accuracy may be inferior to those of a commercial system. still, it is worth noting that such alternatives are also available on the market for a range of motion assessment. smartphone apps with the advancements in smartphone technology, smartphone apps have emerged as a potentially more affordable option for assessing joint range of motion and identifying joint asymmetry (27, 28). newer models of smartphones are usually equipped with highperformance motion sensors such as gyroscopes, accelerometers and magnetometers which could potentially be used to assess joint mobility (54). compared to most previously mentioned methods, smartphone apps are usually cost-effective and easily available to most people who own a smartphone, making them a convenient option for practitioners. there are numerous free or low-cost apps for a range of motion assessment that smartphone users can easily download; however, the accuracy of measurements taken by these apps can differ significantly from one app to another (17, 45, 58, 61). some apps that have already been studied for their reliability and validity in measuring range of motion include: romcam is a relatively new app that utilizes 2d web cameras and openpose (github, san francisco, california, usa) free library based on machine learning models in order to track and detect the 2d key points of human anatomical segments (60). although initial studies have seemedj 2023, vol 7, no 1 computer vision for range of motion 61 southeastern european medical journal, 2023; 7(1) found good reliability and validity of this app for pose-estimation assessment, more research is needed to fully evaluate the scope of its potential for a range of motion assessment in sports and rehabilitation (60). iphone® compass app (apple inc., california, usa). a study by furness et al. (2018) examined the potential of using the compass app, which is pre-integrated into the iphone’s basic software package, to measure the thoracic rotation range of motion (17). the assessment was performed by positioning an iphone firmly against the t1-t2 levels of the participant’s back during active thoracic rotation. results showed good to excellent inter-rater reliability (icc=0.72–0.89) and concurrent validity (r=0.835, p<0.001) compared to the goniometer (17). goniometer pro (digiflex labs, skien, norway) is an app designed to act as a dual-axis goniometer and bubble inclinometer. based on previous research, this app showed goodexcellent reliability (icc=0.79–0.82) in measuring active wrist range of motion (58) and shoulder range of motion (11), as well as good concurrent validity (r≥0.80) compared to a universal goniometer (58) and inclinometer (71). another study also showed excellent reliability of this app (icc=0.995–1.000) in measuring angular changes that normally happen during the range of motion assessment (71). most apps for a range of motion assessment rely on the smartphone camera or built-in motion sensors (i.e. accelerometer, inclinometer, etc.) for data collection (54). therefore, the limitations of using apps to assess rom greatly depend on the smartphone model; for instance, older smartphones may not have the technology to accurately measure the range of motion. moreover, the battery capacity of older models may be degraded, which could result in the phone shutting down during or prior to data collection, and lead to the loss of important information (58). additionally, smartphone sensors are companymanufactured and cannot be calibrated by the user, which can also be problematic for older smartphone models that do not have welldeveloped sensor technology (17). practitioners should also be aware that the accuracy of measurements can originate from the app itself or the experience of the rater; therefore, while smartphone apps offer a convenient and accessible option for joint rom assessment, these limitations should be taken into consideration when interpreting the results. discussion with the rapid development of technology, new solutions for a range of motion assessment are introduced regularly. this study aimed to offer insight into novel computer vision-based systems that can potentially be used for a range of motion assessments in sports and rehabilitation fields. as traditional tools for a range of motion assessment (i.e. goniometer and inclinometer) are often limited in their accuracy due to the subjective nature of the assessment (1), novel computer vision-based systems can provide more objective and precise measurements, as well as more detailed information about joint kinematics (2, 4, 11). these systems include motion-capture cameras (i.e. 2d and 3d cameras), rgb-depth cameras (i.e. kinect), commercial software systems and smartphone apps. while 3d systems (i.e. vicon and qualisys) are unmatched in their precision and are widely considered the golden standard for kinematic analysis, their high cost and robust design limit their practicality and widespread use in sports and rehabilitation (4, 63). therefore, rgb-depth camera systems, commercial software systems and smartphone apps pose a much more feasible solution for assessing a range of motion in a practical setting (2). based on studies evaluating the validity of these novel systems compared to what is considered the golden standard (i.e. 3d motion capture system or goniometer), rgb-depth cameras (i.e. kinect, microsoft corp., redmond, wa, usa) stand out as the most promising option for a range of motion assessment in practical settings, especially when paired with commercial software systems or smartphone apps (39, 72). seemedj 2023, vol 7, no 1 computer vision for range of motion 62 southeastern european medical journal, 2023; 7(1) more specifically, kinect paired with mira software (mira rehab ltd., london, uk) shows excellent validity of shoulder range of motion measurements (r=0.96–0.99) compared to vicon (72). in addition to that, smartphone apps such as goniometer pro (digiflex labs) and compass app (apple inc., california, united states) also show good validity results (r≥0.70) for a certain range of motion measurements (i.e. shoulder and wrist rom) (11, 17). however, further research is needed to better assess the accuracy of these systems in automatic pose estimation, which is crucial for measuring the range of motion (5). the major benefit of rgb-depth camera systems is that they can easily be mounted in any environment without requiring highly specialized knowledge to operate them (64). moreover, they can be paired with smartphone apps (i.e. goniometer pro) or commerciallyavailable and open-source software systems in order to obtain more precise and field-specific information (39, 72). a possible practical application of these systems in sports and rehabilitation includes remote and real-time monitoring of a range of motion changes during exercise. in sports, this can help trainers track various performance parameters that are important for movement efficiency and injury prevention. while in rehabilitation, this can enable a more objective assessment of the range of motion, as well as facilitate remote sessions for clients who cannot attend in-person appointments. however, further research is required to better assess the possibilities of using these systems in such a way. conclusions overall, motion capture systems based on computer vision have the potential to significantly improve the range of motion assessment compared to traditional methods such as goniometry, inclinometry and visual estimation. these systems provide more objective and accurate measurements of the range of motion and offer the possibility of realtime or remote feedback, as well as tracking changes in joint kinematics over time. however, as each system mentioned in this review has its advantages and limitations, it is difficult to determine which system could best replace traditional methods used for a range of motion assessment. as these systems continue to develop and become more accessible to the general public, they may become the standard for assessing a range of motion in the future. however, more research is needed to fully assess their accuracy and potential before implementing them in the field of sports and rehabilitation. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. adams ps, keyserling wm. three methods for measuring range of motion while wearing protective clothing: a comparative study. int j ind ergon. 1993; 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(mišković a, degmečić d. suicidality in depressive patients. seemedj 2018; 2(1); 20-28) seemedj 2018, vol 2, no. 1 suicidality in depressive patients 21 southeastern european medical journal, 2018; 2(1) introduction according to available data from the registers of the croatian institute of public health, one can see that in 2013 there were more than forty thousand hospitalizations associated with mental disorders registered, while depressive disorders were the cause of more than 13% of hospitalizations and the cause of 11% of day hospital treatments used due to mental disorders. (1). according to the criminal code of the republic of croatia, suicide as a criminal act is prosecuted in the case when the suicide is encouraged by another person. the criminal processing is conducted exclusively on a person who encouraged another person to the attempts to commit suicide. (2) a number of risk factors that can lead to suicide and attempts to commit suicide have been identified, and. affective disorders or personality disorders are mostly addressed (3). there is a specific sequence of behavior which occurs before attempting suicide, i.e. the pre-suicidal behavior syndrome and appeal-phenomenon which are often neglected and are often ignored (4). ringel defined the phenomena of parasuicidal and presuicidal behavior, and thus gave an insight into the events of psychopathological changes that lead up to suicide attempts. the first phase, which ringel termed “insufficiency and narrowing”, is the period in which fear and sadness arise and are present to an extent which limits the individual in performing everyday activities. from such feelings, the second phase is developed, a phase of aggression, when an individual directs aggression towards him/ herself because of isolation from the environment. fantasy about suicide or the escape phase constitutes the third phase, which develops from a relief mechanism into concrete ideas by which the suicidal person seeks to escape to a better world. at that stage, he/she considers the techniques and methods of committing suicide and increasingly thinks of oneself as the late one. the fourth phase, the phase of mental anesthesia, is the period in which dissociation of the individual’s personality is noticed. one person performs the tasks of daily life, and the other is preparing for suicide (5). the rate of committing suicide in the general population is 20 : 1, while in clinical cases, in individuals with depressive episodes, the rate is much higher, ranging from 5-10 : 1(6). risk factors for committing suicide that most commonly occur in individuals suffering from depressive disorder are most commonly associated with symptoms of the disease or with specific situations, features of personality, the specifics of family medical history and similar. the groups affected by these disorders are melancholic depressive individuals with a high level of selfcriticism and low level of self-esteem, as well as adolescents and older adults (7). in the republic of croatia, in the period from 2000 to 2014, the suicide rate dropped from 20.9 to 16.3 per 100000 of citizens, but then the suicide rate grew to 722 (17 committed suicides per 100000 persons) in 2015. the ratio of men and women in the observed period ranges from 2.2 3.7 : 1. the rate of suicide in the general population grows with age, with the highest rates being found in the population older than 65. the most common way of committing suicide in both sexes within the general population is hanging (8). beside the risk factors, that there are also protective factors that reduce the possibility of suicide. this includes family and social support, pregnancy, postpartum period, a larger number of children in the family and strong religious beliefs. more protective factors are the care for health and regular physical check-ups, regular application of therapies and optimal physical activity (9). the aim of the present study is examine the incidence of suicides in patients with depressive disorders who were hospitalized at the psychiatric clinic of the clinical hospital centre osijek during 2015 and 2016, and to determine the relationship between sociality and parameters measured in this research. methods this research included 325 depressive patients hospitalized in the psychiatric clinic of the clinical hospital centre osijek during 2015 and 2016. data were collected from the medical records of patients diagnosed with depressive disorders. to collect data the authors used a questionnaire made for the purpose of this research with the following information: age, seemedj 2018, vol 2, no. 1 suicidality in depressive patients 22 southeastern european medical journal, 2018; 2(1) gender, employment status, marital status, qualifications, number of children, the existence of suicide attempts or repeated suicide attempts, motivation for attempted suicide, whether they live alone or with their families, psychiatric heredity, comorbidity. all data were collected from 15 january 2017 to 1 may 2017 and recorded so as not to reveal the identity of the patient.attempts, the way in which the suicide was attempted, number of hospitalizations and treatment duration in years, number of suicide. statistical analysis categorical data are presented with absolute and relative frequencies. numerical data are described by the median and the boundaries of the interquartile range. the differences in categorical variables were tested by the chisquared test and, if necessary, by fisher's exact test. the normality of distribution of numerical variables was tested by the shapiro-wilk test. the differences between the numerical variables between the two independent groups were tested by mann-whitney in the test (23.24). all p values are two-sided. the significance level (medcalc software bvba, ostend, belgium; http://www.medcalc.org; 2014) was used is set to alpha = 0.05. for statistical analysis, the medcalc statistical software version 14.12.0. results out of the total number of respondents (n=325), 119 (36.6%) patients exhibited a suicidal behavior pattern in the past, significantly more in 2015 than in 2016 (fisher’s exact test, p<0,001), at present the suicidal behavior was present at 134 patients, significantly more in 2015 than in 2016 (fisher’s exact test, p=0.04). eighty (24.6%) respondents had attempted suicide. from 80 (24.6%) respondents who had attempted suicide, 33 (41.3%) were men and 47 (58.8%) were women (table 1). table 1. suicidal behavior pattern in the past and in the present, suicide attempt according to the sex of respondents number (%) of respondents p* 2015 2016 total suicidal behavior pattern in the past yes 71 (47.3) 48 (27.4) 119 (36.6) < 0.001 no 79 (52.7) 127 (72.6) 206 (63.4) suicidal behavior pattern in the present yes 71 (47.3) 63 (36) 134 (41.2) 0.04 no 79 (52.7) 112 (64) 191 (58.8) suicide attempt yes 38 (25.3) 42 (24) 80 (24.6) 0.79 no 112 (74.7) 133 (76) 245 (75.4) total 150 (100) 175 (100) 325 (100) suicide attempt according to sex male 17 (44.7) 16 (38.1) 33 (41.3) 0.65 female 21 (55.3) 26 (61.9) 47 (58.8) total 38 (100) 42 (100) 80 (100) * fisher's exact test there were no significant differences in 2015 and 2016 in terms of the number of suicide attempts (table 2). seemedj 2018, vol 2, no. 1 suicidality in depressive patients 23 southeastern european medical journal, 2018; 2(1) table 2. number of suicide attempts in years 2015 and 2016 number of suicide attempts number (%) of respondents p* 2015 2016 total one 23 (60.5) 30 (71.4) 53 (66.3) 0.82 two 9 (23.7) 7 (16.7) 16 (20) three 1 (2.6) 1 (2.4) 2 (2.5) four 0 (0) 1 (2.4) 1 (1.3) five 1 (2.6) 1 (2.4) 2 (2.5) >five 4 (10.5) 2 (4.8) 6 (7.5) total 38 (100) 42 (100) 80 (100) * fisher's exact test place of residence, marital status, the basic diagnosis and treatment duration do not seem to be connected with the respondent’s attempted suicide. in terms of number of suicide attempts, there was a statistically significant difference between subjects who had had suicidal ideas in the past or who presently had such ideas on the one side and those who never had such ideas on the other, with the former subjects being the ones who attempted suicide more often (fisher’s exact test, p < 0.001) (table 3). table 3. respondents according to the basic diagnosis, suicidal behavior pattern and suicide attempt number (%) of respondents according to suicide attempts p* no yes total location rural 128 (52.2) 43 (53.8) 171 (52.6) 0.89 urban 117 (47.8) 37 (46.3) 154 (47.4) marital status married 159 (64.9) 48 (60) 207 (63.7) 0.39 single 42 (17.1) 11 (13.8) 53 (16.3) divorced 40 (16.3) 19 (23.8) 59 (18.2) in a relationship 4 (1.6) 2 (2.5) 6 (1.8) the basic diagnosis depressive episode (f32) 75 (30.6) 29 (36.3) 104 (32) 0.41 recurrent depressive disorder (f33) 170 (69.4) 51 (63.8) 221 (68) treatment duration 1 year 32 (13.1) 11 (13.8) 43 (13.2) 0.66 2 years 13 (5.3) 4 (5) 17 (5.2) 3 years 9 (3.7) 6 (7.5) 15 (4.6) 4 years 5 (2) 0 5 (1.5) 5 years 9 (3.7) 3 (3.8) 12 (3.7) >5 years 177 (72.2) 56 (70) 233 (71.7) suicidal ideas in the past yes 51 (20.8) 68 (85) 119 (36,6) < 0.001 no 194 (79.2) 12 (15) 206 (63.4) suicidal ideas in the present yes 74 (30.2) 60 (75) 134 (41.2) < 0.001 no 171 (69.8) 20 (25) 191 (58.8) total 245 (100) 80 (100) 325 (100) * fisher's exact test seemedj 2018, vol 2, no. 1 suicidality in depressive patients 24 southeastern european medical journal, 2018; 2(1) the median age of the respondents who tried to commit suicide was 53 years of age (interquartile range from 43 to 60 years). the age of respondents was from 17 to 76 years, similar to those who have not tried suicide (table 4). table 4. age of the respondents according to suicide attempt the median age (interquartile range) of the respondents who tried to commit suicide p* no yes total age of the respondents [years] 55 (49 62) 53 (43 60) 54 (46 61) 0.05 *mann whitney u test the median age of respondents with one single attempted suicide was 54 (interquartile range from 45 to 60 years old), while the median of respondents with more than one attempt was 51 (interquartile range from 37 to 62 years) without statistically significant differences between those two groups (table 5). table 5. age of the respondents with one single attempted suicide and respondents with more than one attempt the median age (interquartile range) of the respondents according to the number of suicide attempts p* one single attempted suicide more than one attempt total age of the respondents [years] 54 (45 60) 51 (37 62) 54 (46 61) 0.28 *mann whitney u test for 38 (11.7%) respondents, family problems were the motive to attempt suicide, interpersonal problems motivated 33 (10.2%) of the respondents, and there were 7 (2.2%) of the respondents without a clear motive. the existence of psychiatric heredity was present in 94 (28.9%) of the respondents, significantly more among respondents who attempt suicide in the 2015 than in 2016 (fisher’s exact test, p < 0.001), and 20 (6.2%) of the respondents had had experience with attempted suicide in their families (table 6). table 6. motive to attempt suicide, psychiatric heredity and attempted suicide in families in years 2015 and 2016 number (%) of respondents p* 2015 2016 total motive to attempt suicide family problems 17 (11.3) 21 (12) 38 (11.7) 0.87 interpersonal problems 19 (12.7) 14 (8) 33 (10.2) 0.20 without a clear motive 2 (1.3) 5 (2.9) 7 (2.2) 0.46 psychiatric heredity yes 59 (39.3) 35 (20) 94 (28.9) <0.001 no 84 (56) 138 (78.9) 222 (68.3) unknown 7 (4.7) 2 (1.1) 9 (2.8) total 150 (100) 175 (100) 325 (100) attempted suicide in family yes 11 (7.4) 9 (5.1) 20 (6.2) 0.49 no 137 (92.6) 166 (94.9) 303 (93.8) total 148 (100) 175 (100) 323 (100) * fisher's exact test seemedj 2018, vol 2, no. 1 suicidality in depressive patients 25 southeastern european medical journal, 2018; 2(1) the existence of psychiatric heredity or suicide attempts in their families does not affect the suicide attempts of the respondents themselves. the number of respondents who attempted suicide and who had positive psychiatric heredity was 53 (66.3%), while 169 (69%) of the respondents did not have any experience with attempting suicide in their families or positive psychiatric heredity (table 7). table 7. respondents according to psychiatric heredity and attempted suicide in the family number (%) of respondents according to suicide attempts p* no yes total psychiatric heredity yes 68 (27.8) 26 (32.5) 94 (28.9) 0.55 no 169 (69) 53 (66.3) 222 (68.3) unknown 8 (3.3) 1 (1.3) 9 (2.8) total 245 (100) 80 (100) 325 (100) attempted suicide in the family yes 16 (6.6) 4 (5.1) 20 (6.2) 0.79 no 228 (93.4) 75 (94.9) 303 (93.8) total 244 (100) 79 (100) 323 (100) * fisher's exact test discussion as the authors stated at the very beginning, affective diseases are most common among persons who commit suicide. the lifetime risk of suicide in patients with depression is 15% (10). it should be emphasized that in patients suffering from depressive disorders, the risk of suicide is twenty times higher than in the general population (11, 12). in present study, 9.2% of respondents indicated deliberate self-harm and suicide attempts as the reasons for their last hospitalization. on the other hand, data from literature show that, out of the total number of respondents, a quarter of them had attempted suicide either recently or in the past, without statistically significant differences according to the sex of the respondents. suicide is three times more common in men, which was not found within our selected patient population. the authors can see from the results that there are no statistically significant differences regarding sex of the person who committed suicide (13). in addition, from the literature authors could see that the ratio of suicide attempts in men and women varied based on sociodemographic characteristics, which indicates that in countries with a higher standard of living there is a higher frequency of suicide attempts in males, while the frequency of suicide attempts among women shows a higher percentage in countries of medium and low living standards (14). in present study, however, there was no statistically significant relationship found between the place of living, so we can conclude that, in the case of present population, the living standard according to the place of living did not have any influence on whether the respondent attempted suicide or not. the lowest rate of attempted suicides was found in married people seemedj 2018, vol 2, no. 1 suicidality in depressive patients 26 southeastern european medical journal, 2018; 2(1) and that single life increases the risk by 2 times. the same applies to divorced and widowed persons, whose risk is also twice as high as the risk found in singles (13). on the other hand, in present study there was no statistically significant relationship between the marital status of the patients or whether they lived alone or in a young society in his /her own family and the intention for committing suicide. published studies show that people suffering from depression with the intent to commit suicide are of the average age of 55 (15). in present population, the average age of respondents who had attempted suicide was 53 years of age, with no significant differences in relation to those who had not attempted suicide, which coincides with data from the literature and is included in their interquartile age range. in present study, we hypothesized that the number of hospitalizations is connected with the attempts of suicide, however, this was not shown to be statistically significant and corresponds to the data from literature, which indicates that the number of psychiatric consultations did not correlate with increased risk of suicide in depressed patients (16). the most common motives that we can find in literature are interpersonal relationships, lack of love in the family, and loss of control over their disease (17). present research has shown that the most common motives for suicidal behavior are family problems and interpersonal reasons. for example, kieholz's scheme of judgement and determination of suicidal tendencies has shown that the presence of suicide(s) in the patient’s immediate family or among other close relatives increases the risk of suicide possibility (18). we can notice how this scheme can be applied to our study because people with suicidal ideas in the past or those with suicidal ideas at the present time attempted suicide more often. numerous studies, including family studies, studies of twins and adoptive researches, associate family communication and the possibility of inheritance of suicidal behavior (reviewed by 19). it is mentioned that the risk for a depressive episode is an independently inherited factor (20). in present research, based on the selected population, the existence of psychiatric heredity or suicide attempts in the family did not affect the attempted suicide of the respondents. in the actual attempted suicide, the most common method of execution of the act involves deliberate self-harm by using drugs/intoxication, which is also the most persistent in the form of suicide attempts, with deliberately intoxication and the effects of alcohol being found much more often in 2015. deliberate self-harm by hanging, strangulation, and asphyxiation showed no significant differences during monitoring period. data from the world health organization show that the methods of attempted suicides are different in certain parts of the world. the authors can distinguish three most common methods of attempted suicides in the world: hanging, poisoning with organophosphates and pesticides, and suicides by firearms. given the fact that croatia is a part of europe, we have compared the obtained data with the rest of europe, where it has been shown that the most common methods of suicide attempts, in the respondents in this study, were deliberate selfharm by using drugs and suicide attempt with a firearm, which coincides with the results obtained in present study (21). summary of the findings of present study are: a) recurrent depressive disorder (f33) was more common in 2016, while depressive episode (f32) was more common in 2015 (out of the total number of patients suffering from a depressive disorder); b) the most common comorbidity diagnoses of the respondents were in the area of mental disorders and disorders of behavior, secondly there were diagnoses in the fields of circulatory system diseases, while the least comorbidity was found in the area of skin and subcutaneous tissue diseases; c) suicidal forms of behavior in terms of suicidal ideas in the past and suicidal ideas in the present were significantly more present in 2015; d) there were no significant statistical differences with regard to gender and age of the respondents in relation to their attempted suicides, e) place of residence, marital status, the basic diagnosis and treatment duration were not found to be connected with whether the respondent attempted suicide or not; e)subjects with suicidal ideas in the past or in the present seemedj 2018, vol 2, no. 1 suicidality in depressive patients 27 southeastern european medical journal, 2018; 2(1) attempted suicide more frequently, to a statistically significant degree, f) the most common motives of suicidal behavior patterns were family problems and interpersonal considerations; g) the existence of psychiatric heredity or attempts of suicide in the family were not found to be connected with suicide attempts of the respondents; h) the number of psychiatric hospitalizations was not found to be connected with suicide attempts of depressed patients. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. croatian institute of public health. department for mental disorders with psychoses registry and committed suicides registry. croatian committed suicide registry available from: http://www.hzjz.hr/sluzbaepidemiologija-zaraznebolesti/odsjek-za-mentalneporemecaje-s-registrom-za-psihozei-registrom-izvrsenih-suicida/. date last accessed: 28 february 2017. 2. criminal code of the republic of croatia. criminal offences against life and limb. official gazette .2014;125/11, 144/12, 56/15, 61/15. 3. hawton k, van heeringen k. suicide. the lancet 2009;373:1372–1381. 4. folnegović-šmalc v, kocijanhercigonja d, barac b. prevencija suicidalnosti. zagreb: multigraf; 2001. pg. 35-43. 5. ringel e. da odbaciš život?: refleksije o suicidu. zagreb: biblioteka ¨oko 3 ujutro¨; 1983. pg. 7-15. 6. mindoljević-drakulić a. suicid fenomenologija i psihodinamika. zagreb: medicinska naklada; 2013. pg. 100-103. 7. kozarić-kovačić d, jendričko t. suicidalnost i depresija. medicus 2004;1:77 – 87. 8. stevanović r, capak k, benjak t. croatian health statistics yearbook 2015. croatian committed suicides registry. croatian institute of public health 2016;289-90. 9. rihmer z. depression and suicidal behaviour. chichester:wileyblackwell publishing. 2011. pg. 53-73. 10. guze sb, robins e. suicide and primary affective disorder. br j psychiatry 1970;117:437-8. 11. harris ec, barraclough b. suicide as an outcome for mental disorders, a meta-analysis. br j psychiatry 1997;170:205-28. 12. chen yw, dilsaver sc. lifetime rates of suicide attempts among subjects with bipolar and unipolar disorders relative to subjects with other axis i disorders. biol psychiatry 1996;39:896-9. 13. novak l, labura d. suicid u mladih i uloga prvostupnika sestrinstva u prevenciji suicida, final paper. zadar: university of zadar, department of health studies. 2016. 14. world health organization. preventing suicide: a global imperative. executive summary. available from: http://www.who.int/mental_health/ suicideprevention/exe_summary_english.p df?ua=1. date last accessed: 21 april 2017. 15. us department of health and human services. office of applied studies, substance abuse and mental health services administration (samhsa). suicidal thoughts, suicide attempts, major depressive episode, and substance use among adults. available from: http://www.samhsa.gov/data/2k6/ suicide/suicide.pdf. date last accessed: 21 april 2017. seemedj 2018, vol 2, no. 1 suicidality in depressive patients 28 southeastern european medical journal, 2018; 2(1) 16. roy a. depressed patients who suicide at their first attempt have had few admissions. depress anxiety 1999; 9:75-7. 17. marčinko d. teorija suicida. pro mente croatica 2003/2004;7:15—16. 18. folnegović-šmalc v, folnegović grošić p, henigsberg n, eds. farmakoterapija depresija. medicus 2004;1:31 – 39. 19. brent da, mann jj. family genetic studies, suicide, and suicidal behavior. am j med genet c semin med menet 2005;133(1):13-24. 20. gershon es. genetics. new york: oxford university press; 1990. 373– 401. 21. ajdacic-gross v, g weiss m, ring m, hepp u, bopp m, eds. methods of suicide: international suicide patterns derived from the who mortality database. available from: http://www.who.int/bulletin/volum es/86/9/07-043489/en/. date last accessed: 21 april 2017. 22. silobrčić-radić m, jelavić m, tomić b, ćorić t,stevanović r, eds. mentalni poremećaji u republici hrvatskoj. zagreb: croatian institute of public health. 2011.33-34. 23. ivanković d. i sur. osnove statističke analize za medicinare. zagreb: medicinski fakultet sveučilišta u zagrebu; 1988. pg. 335-48 24. marušić m. i sur. uvod u znanstveni rad u medicini. 4. izd. udžbenik. zagreb: medicinska naklada; 2008. pg. 3290. seemedj 2017, vol 1, no. 1 interventions of health visitors in making a decision about breastfeeding 130 southeastern european medical journal, vol 1, 2017. interventions of health visitors in making a decision about breastfeeding 1 štefica mikšić1, zdenka konjarik2, mirjana kralj-vasilj1, brankica juranić1, dragica pavlović1 1 josip juraj strossmayer university of osijek faculty of medicine osijek, cara hadrijana 10/e, osijek, croatia 2 specialist pediatric outclinic, đakovo; croatia corresponding author: štefica mikšić, m.sc. in nursing steficamiksic@gmail.com introduction preparing a pregnant woman and family members to accept positive attitudes about received: march 22, 2017; revised version accepted: may 25, 2017; published: may 26. 2017 keywords: promotion of breastfeeding, visiting nurse breastfeeding begins with educating and sensitizing the local community. breastfeeding is good for the family, and society as a whole. the world health organization (who) and unicef are focused on the application of natural abstract aim: the aim of this paper was to establish a link between the promotion of breastfeeding and the interventions of the visiting nurse. methods: the study was conducted in 2012 in the city of đakovo and the surrounding area. the questionnaire was filled out by mothers after leaving the maternity ward, during the first visit of the community health nurse within the first seven days after the labor. results: from a total of 154 parturient women who filled in a survey after they had left the hospital, the decision about breastfeeding before the pregnancy was made by 58.4% of examinees. primiparas from the city were older than primiparas from the surrounding rural area (p<0.001). mothers received the majority of information about the proper placement of the child on the chest after birth from nurses in the maternity hospital (56.3% of the examinees). the study showed that 57.9% of the examinees had the support of their husband during lactation. the visiting nurses’ first visit was within seven days after delivery at the home of 121 (78.6%) examinees, showing good awareness and collaboration between secondary and primary health care. conclusion: this research has confirmed that breastfeeding promotion and nursing interventions have a major impact on breastfeeding. (mikšić š, konjarik z, kralj-vasilj m, juranić, b, pavlović d. interventions of health visitors in making a decision about breastfeeding. seemedj 2017;1(1);130-135) seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 131 southeastern european medical journal, vol 1, 2017. nutrition to ensure the health of the child and mother (1). there is a need for intervention by gynecologists, maternity clinics, pediatricians, patrons, family physicians, nurses working in ambulances and pharmacists. breastfeeding should be a choice, not a behavior that is imposed on the mother (2). it implies feeding the baby on the mother's chest, because breast milk is the best baby food for newborns (3). the length of breastfeeding is an indicator of the interest, time and energy that a mother intends to invest in her child's development. rooming-in is a 24-hour stay of the mother and the baby with the goal of starting breastfeeding as early as possible. during breastfeeding, a mother transfers to her child feelings of warmth, safety and protection (4). research has shown that early skin contact, besides stimulating breastfeeding, also affects the rhythm of sleep and sleepiness, and stimulates the motor and neurological behaviors of newborns after delivery. these positive effects were also observed in premature babies and term children (5). mothers who breastfeed for a longer time spend more time with their child and achieve the interaction that favors their child’s cognitive and intellectual development (6). according to the estimation of the health nurse, at least one or several patron intervention visits are arranged for the protection and care of the pregnant woman. during child monitoring, the mother should be instructed that she should be available to the child for unlimited breastfeeding (7). this process creates the proper bonding of mother and child and affects a different pattern of the mother’s behavior. it is a special psychophysical stimulus for a mother and a child that improves the cognitive development of the child. mother's milk, with its composition, fully meets the needs of the baby for food and fluids during the first six months of the baby's life, and as a dairy meal can be delivered even after the first year. the effects of the optimal composition of the mother's milk and the influence of certain ingredients stimulate the development of the baby's brain, and are not produced by other milk types. establishing the importance of breastfeeding encourages an environment in which the mother has the support of family members, and the promotion of breastfeeding is carried out by health care professionals. the most important supportive role to the mother in breastfeeding belongs to the father of the child, who must be familiar with the benefits of breastfeeding and have a positive attitude towards it (8). the aim of this study was to establish a link between the promotion of breastfeeding and the interventions of visiting nurses. materials and methods the research was conducted in the town of đakovo and its surroundings, during the first house call made by health visitors after the mother had been discharged from the hospital. from a total of 202 babies born during the survey, there were 154 mothers who were breastfeeding their babies and were included in the research. the research was carried out by means of an anonymous survey. other issues addressed concern the relationship between breastfeeding and the way of birth in these pregnancies, the duration of pregnancy, education on breastfeeding and support during breastfeeding. afterwards, questions were asked related to previous births and the length of breastfeeding of children. statistical analysis the frequency distributions for investigated variables were described by descriptive statistical methods. the kolmogorov–smirnov test was used to analyze the normal distribution of the variables. the mean values for the continuous variables have been expressed as the arithmetic mean and as the standard deviation for normally distributed variables, and for variables that do not have a normal distribution, as median and range. the nominal data have been expressed through the frequency distribution both in groups and its content. χ²-test and fisher exact test have been used to determine the differences between the two independent samples. the significance level α = 0.05 was selected to evaluate the importance of the obtained results. seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 132 southeastern european medical journal, vol 1, 2017. *fisher exact test figure 1. division of examinees (%) according to the person who taught them how to breastfeed table 1. breastfeeding signs regarding the time when the decision on breastfeeding was decided breastfeeding signs time when the decision was made total n (%) p* before the pregnancy n (%) during the pregnancy n (%) after the delivery n (%) does not want to breastfeed n (%) bmi before pregnancy 21.68 3.18 1.20 24.42 5.58 0.001 mouth wide open, round cheeks, baby is close to the mother's body, baby is calm and relaxed, the baby holds the breast with its fingers by itself and lets go of the breast by itself after it has been fed 64 (71.1) 37 (72.5) 3 (27.3) 2 (100) 104 (67.5) baby's mouth is not wide open, cheeks are sunken, breasts are painful and sore, there is only some contact between the mother and the baby 14 (15.6) 7 (13.7) 3 (27.3) 24 (15.6) baby's mouth is not wide open, cheeks are sunken, breasts are painful and sore – there are rhagades, there is no contact between the mother and the baby 3 (3.3) 3 (5.9) 6 (3.9) the mother is tense and in an awkward position, the baby is restless and cries, the baby can't take the breast by itself, breastfeeding is painful and short 2 (3.9) 1 (9.1) 3 (1.9) the baby does not suckle 9 (10) 2 (3.9) 4 (36.4) 17 (11) total 90 (100) 51 (100) 11 (100) 2 (100) 154 (100) seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 133 southeastern european medical journal, vol 1, 2017. results there were 154 women who took part in the research, of which 79 (51.3%) live in rural areas, and 75 (48.7%) in the city. the median age in which mothers had their first baby is 26 for the women living in town, and 23 for those living in rural areas (mann whitney u test, p<0.001). mothers who live in the town have breastfed their first baby longer, whereas mothers who live in rural areas have breastfed their second baby longer. most of the examinees received the information on breastfeeding from the nurses in the maternity hospital and from the health visitor (figure 1). during breastfeeding, 88 (57.9%) of the examinees were supported by their husbands, 56 (36.8%) by their extended family and 8 (5.3%) and by other services (i.e. health care service, bsg breastfeeding support group). evaluation of examinees’ breastfeeding has shown much better results in the cases of those who made the decision to breastfeed before or during their pregnancy. the lowest incidence of breastfeeding is in the group of examinees who made the decision to breastfeed after delivery (table 1). it was found that a group of women who gave birth to one, two and three children statistically differ significantly in the number of months of breastfeeding (f (2.133) = 12.14, p <0.01, η2 = 0.154). in a post-hoc comparison (bonferroni), a statistically significant difference was found between women who had a first child (m = 1.66, sd = 4.37) and women who had a second child (m = 7.34, sd = 8.87). in order to determine whether there was a difference between women who had a second or third child, a t-test was carried out. it was found that a group of women who gave birth to two and three children statistically differ significantly in the number of months breastfeeding another child (t = 6.45, df = 65, p <0.01). there was a statistically significant difference between women who had a second child (m = 1.02, sd = 0.15) and women who had a third child (m = 6.25, sd = 5.34). discussion by educating pregnant women and family members, the adoption of breastfeeding as the only way to properly feed the baby is accomplished. interventions of visiting nurses are conducted through individual and group work (9). mothers are more likely to make a decision about breastfeeding if they are well informed about the benefits of breastfeeding and if they have family, social, and especially their husbands’ support. while 57.9% of examinees had the support of the husband during breastfeeding, only 5.3% had the support of outside groups, suggesting that breastfeeding support groups should be expanded because the mother is the best educator. studies have shown that a woman whose husband does not support breastfeeding stops earlier or doesn't even start breastfeeding (8). the first visit of the nurses within 7 days after the birth occurred in 78.6% of the cases, indicating good communication and cooperation between the hospital and the patrons' service. it is very important for a mother to know where and how she can get all the information needed after birth. the visiting nurse must be familiar with the physiology of lactation to promptly detect potential problems with breastfeeding and to arrange, together with the new mothers, a health care and intervention plan for solving any identified problems. a significantly better estimation of breastfeeding was observed among those who decided to breastfeed before pregnancy (table 1). mothers with a clear plan for the duration of breastfeeding usually follow through with it, while hesitant mothers generally breastfeed for a shorter amount of time (10). mother and child should master the breastfeeding technique, and the mother should be instructed to inflate the baby, which means that the frequency and length of breastfeeding depend on the child's needs and signs. signs that breastfeeding is progressing or not progressing depend on the emplacement of mothers, breastfeeding and signs of the transfer of the milk (11). our research has shown that previous experience is an important factor because the respondents’ second child nursed longer than the third child. seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 134 southeastern european medical journal, vol 1, 2017. respondents of the study have consistently demonstrated that women who gave birth to more than one child have a higher breastfeeding efficacy than women who gave birth to one child (12, 13, 14). a positive experience of breastfeeding can improve the mother's confidence in breastfeeding, and a negative experience of breastfeeding can reduce it. other studies have shown that 30-57% of mothers have problems with breastfeeding in the early postpartum age, where insufficient milk is reported as the most common problem and the primary cause of early termination of breastfeeding (15, 16). a well-educated mother is more prepared for problems that may occur during lactation, and she has a different pattern of behavior. studies have shown that the mother who spends more time with her child is aware of the benefits of her milk for the growth and development of the baby, and also that in a short period of time the child developed a good grip on the breasts (17,18) (table 1). children have both the need for food as well as the need for touch, gentleness and love that only the mother can provide in the first days of life. pinard's aphorism says "there is no replacement for mother's milk and her heart" (19). who, unicef and the american pediatric academy (aap) plead for breastfeeding only up to 6 months of age and then a continuation of breastfeeding along with the right meal for frozen foods for at least one year (20) or two years (21), and even longer if so wanted by both mother and child. conclusion this research has shown that the most important breastfeeding factors are the mother's decision before delivery and an early visit by a nurse after coming home from the maternity ward. the visiting nurse is the first health care worker to start training pregnant women during pregnancy about breastfeeding and continues after the mother's return from the maternity ward. also, the visiting nurse’s approach to the mother and newborn is of great importance, and their imparted knowledge is the basis for the development of appropriate skills (22) and attitudes so that habits and behavior changes will be adopted in order to preserve and improve the health of pregnant women, new mothers and children in the wider community. acknowledgments the authors gratefully acknowledge the health visitors who made possible the conduct of the research and all the examinees who participated in the research. this paper was presented as a master thesis at the professional study in nursing, faculty of medicine osijek, josip juraj strossmayer university of osijek, june 12, 2012 disclosures funding: no specific funding was received for this study. competing interests: none to declare. references 1. pavičić-bošnjak a, grgurić j. dojenje: ne samo nutritivni, već i razvojni čimbenik. hrvatski zavod za javno zdravstvo; odsjek za neonatologiju, klinika za ženske bolesti i porode, klinička bolnica „merkur“, zagreb 2005. vol.1, br.2. http://www.hcjz.hr/old/clanak.php?id=1247 9. datum pristupa: 2.05.2017. 2. ajduković m, kregar k, laklija m. teorija privrženosti i suvremeni socijalni rad. ljetopis socijalnog rada 2007;14:59-92. 3. hamosh m. bioactive factors in human milk. pediatric clinics of north american 2001;48(1):69-73. 4. mziss. dojenje dar životu. stega tisak d.o.o., zagreb.2007. 5. mortensen el, michaelsen kf, sanders s, reinish jm. the association between duration of breastfeeding and adult intelligence. jama 2002;287:2365-71. 6. priručnik za zdravstvene djelatnike „dojenje“. unicef, 1999. 7. konjarik z. primarna zdravstvena zaštita djece i promicanje dojenja. u: grgurić, j, seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 135 southeastern european medical journal, vol 1, 2017. pavičićbošnjak a, ur. program promicanja dojenja u republici hrvatskoj. zagreb: unicef ureda za hrvatsku, 2007; 82-89. 8. batinica m, grgurić j, lipovac d, vinceković v, batinica r, turčinov e. važnost očeve potpore pri dojenju. pediatr croat 2001; 45: 201-7. 9. hegeduš-jungvirth m, krčmar n, srnec d. rezultati promicanja dojenja u međimurju. pediatr croat 2000;44:91-4. 10. pavičić-bošnjak a, grgurić j. osobine majki članica grupa za potporu dojenja i njihov utjecaj na trajanje dojenja. paediatr croat 2007;51: 89-5. 11. de jager e, skouteris h, broadbent j, amir l, mellor k: psychosocial correlates of exclusive breastfeeding: a systematic review. midwifery 2013; 29: 506-18. 12. znakovi dobrog pozicioniranja, pripajanja i djelotvorno/nedjelotvorno dojenje dostupno na: http://www.husd.hr/savjetnice/smjernice /7-prehrana-dojenceta-i-malog-djetetastandardne-preporuke-za-eu. date of access 2.05.2017. 13. pavicic bosnjak a, stanojevic m, glavas e: odnos između samoefikasnosti dojenja i teškoća pri dojenju tijekom boravka u rodilištu. paediatr croat 2014;58:88-94. 14. dennis cl: the breastfeeding self-efficacy scale: psychometric assessment of the short form. j obstet gynecol neonatal nurs 2003;32:734-44. 15. aluş tokat m, okumuş h, dennis cl: translation and psychometric assessment of the breast-feeding self-efficacy scaleshort form among pregnant and postnatal women in turkey. midwifery 2010;26:101-8. 16. gatti l: maternal perceptions of insufficient milk supply in breastfeeding. j nurs scholarsh 2008;40:355-63. 17. pavicic bosnjak a, stanojevic m, glavas e: odnos između samoefikasnosti dojenja i teškoća pri dojenju tijekom boravka u rodilištu. paediatr croat 2014;58:88-94. 18. joshi a, amadi c, meza j, aguire t, wilhelm s: evaluation of a computer-based bilingual breastfeeding educational program on breastfeeding knowledge, self-efficacy and intent to breastfeed among rural hispanic women. int j med inform 2016;91:10-9. 19. gjurić g. prehrana i poremećaji stanja uhranjenosti. u: mardešić d, ur. pedijatrija, 5 izd. zagreb: školska knjiga, 1991;213-65. 20. american academy of pediatrics. breastfeeding and the use of human milk. pediatrics 2005;115:496-506. 21. who. global strategy for infant and young child feeding. geneva: who; 2003. 22. samardžija čor i, zakanj z. utjecaj patronažne sestre na dojenje, acta clinica croatica, 2014;53(2):204-9. seemedj 2019, vol 3, no. 1 information security and privacy awareness 21 southeastern european medical journal, 2019; 3(1) original article awareness about information security and privacy among healthcare employees 1 kresimir solic 1, mateo plesa 1, tena velki 2, kresimir nenadic 3 1 department of medical statistics and medical informatics, faculty of medicine, josip juraj strossmayer university of osijek 2 department of social sciences, faculty of education, josip juraj strossmayer university of osijek 3 department of software engineering, faculty of electrical engineering, computer science and information technology, josip juraj strossmayer university of osijek corresponding author: kresimir solic, kresimir.solic@mefos.hr abstract aim: the aim of this study was to analyze healthcare employees’ knowledge of information security and potentially risky behavior on the internet considering demographic parameters and in comparison with the standardized behavioral norms among internet users in croatia. methods: the study was conducted as a cross-sectional study. healthcare employees from three hospitals in different geographical areas (osijek, pula and zagreb) were included in this study. the validated uisaq (users’ information security awareness questionnaire) was used for data collection. the questionnaire contains 33 questions, grouped in two scales and six subscales, and participants were self-evaluated using likert scale. the time period of data collection was the summer of 2017. results: surveyed healthcare employees show significantly less risky behavior and overall better knowledge than the average internet user in croatia. female participants display online behavior that is less risky than that of the male participants; participants with a university degree are better at pc maintenance, while participants with a high school diploma are more skeptical in regard to loss of personal or professional data. older people are significantly more careful and lend their access data to other colleagues at work less often. conclusion: healthcare employees included in this study display partially better results than the average internet users in croatia when it comes to their knowledge and potentially risky online behavior. however, their average estimations are only partially better than referent estimations and their scores are not very high, especially when it comes to their awareness measured in the “security in communications” and “secured data” subscales. as there is high risk of losing data because of the nature of business protocols, healthcare employees need more education and training in order for their awareness regarding the importance of information security and privacy to increase. (solic k, plesa m, velki t, nenadic k. awareness about information security and privacy among healthcare employees. seemedj 2019; 3(1); 21-28) received: november 6, 2018; revised version accepted: may 7, 2019; published: may 31, 2019 keywords: information security, privacy protection, risky behavior, internet, uisaq mailto:kresimir.solic@mefos.hr seemedj 2019, vol 3, no. 1 information security and privacy awareness 22 southeastern european medical journal, 2019; 3(1) introduction since the internet has become an integral part of human life, more and more opportunities are being created, both in the positive sense for the advancement of technology and communication between people as well as in the negative sense, which refers to the existence of risks regarding personal security and privacy. therefore, there exists a great need to protect personal data in order to reduce the risk of theft of information from users of all age groups, from the youngest to the oldest. new services on the internet (applications, electronic healthcare, shopping, etc.) that are becoming increasingly necessary and involve more and more users require users to disclose some of their personal information. with this potential risk increasing and seeing as numerous users are ignorant when it comes to the information and communication systems involved, they accept imposed rules and readily start using new services as soon as they appear in the digital market. since previous research has shown that a person as an information system user may be the most critical security element in said system (1 4), the issue of privacy and user protection will most likely never be solved, even though program security, security procedures and backup automation are at a high level. it is certain that the above is not enough to fully protect the user. responsibility and conscientious use of internet services by the user are also required. therefore, reducing said risk is possible, and one of the best ways of doing so is increasing user awareness by educating them about the various types of unwanted events like frauds and privacy loss on the internet. for example, installing additional apps or divulging a small piece of personal data may ultimately result in financial or other, less significant loss, which was by no means the intention of the user who installed or provided said information (5). in general, data protection, not just on the internet, is carried out in order to prevent data theft or data manipulation. there are two reasons for protecting electronic data: the possibility of their loss and the possibility of unauthorized use of data by an unreliable person with malicious intent. there are several ways to protect data, and the most common one is the use of antivirus programs that protect your computer’s operating system from different kinds of malware. before using the computer’s operating system, it is useful to update both the antivirus program and its virus definitions to secure the personal data stored on the computer. malicious people who want to cause harm to computer users and software or operating system manufacturers tend to do so in order to prove that manufacturers did not create the application, program or operating system with sufficient protection mechanisms. malicious people do not benefit greatly from developing and producing viruses and other types of malware (6, 7). healthcare employees are users and integral parts of a hospital’s information and communication system. the hospital information system (his) is a unique information system within a hospital that combines medical and non-medical data created at various hospital departments for a better and more effective way of exchanging information and more successful way of communicating with patients (8). modernizing medicine with accessible information technology in patient management systems provides many benefits, but it is possible to manipulate and abuse the privacy. patient’s personal data availability is important to medical staff in order to provide better medical care, healthcare and treatment (9). the protection of personal data, in particular data relating to the health of persons, is primarily carried out with the aim of protecting the right to privacy of personal and family life, which is one of the personal rights protected by our legislation. similar empirical studies were conducted on the subject using the uisaq, but they focused on other groups of internet users (10 12). the latest study was conducted at the national level as seemedj 2019, vol 3, no. 1 information security and privacy awareness 23 southeastern european medical journal, 2019; 3(1) part of the eu project under agreement number inea/cef/ict/a2015/115320. those results are used as referent values for comparison with results of this study (13). therefore, the aim of this study was to analyze knowledge on information security and potentially risky behavior on the internet among healthcare employees considering demographic parameters and in comparison with the standardized behavioral norms among internet users in croatia. methods the authors used the validated users’ information security awareness questionnaire (uisaq) for data collection in three croatian hospitals located in different geographical areas: osijek, pula and zagreb. the data were collected during the summer of 2017. the study was conducted as a cross-sectional study. the uisaq has two major scales with three subscales each; each subscale contains five or six items (questions). associated abbreviations are used in the subsequent text and tables: • potentially risky behavior (prb; k = 17) o usual behavior (ub; k = 6) o personal computer maintenance (pcm; k = 6) o access data lending (adl; k = 5) • knowledge and awareness (ka; k = 16) o security in communications (sc; k = 5) o secured data (sd; k = 5) o backup quality (bq; k = 6) these subscales describe the user’s behavior, knowledge and awareness (5, 11). participants were asked to estimate how much they agree with a statement on a 5 point likert type scale, where five means excellent, from the aspect of information security. at the end of the uisaq, two additional questions about behavioral security of users were given, as well as a section for the provision of demographic data. statistical analysis the statistical software tool medcalc 14.12.0 was used for statistical analysis in this paper. statistical significance, when comparing differences in estimations among groups, was defined as p < 0.05 using student’s t-test and one way anova with post hoc scheffé test. correlations with age were tested using the spearman’s rank correlation test. results the surveyed healthcare employees were 38.5 ± 11.3 (x ± sd) years old, mostly female (83.8%, p < 0.001, chi-square test) and mostly with a high school diploma (94.1%, p < 0.001, chi-square test). average estimation in the “usual behavior” subscale (student’s t-test, p = 0.03) was significantly higher for women than for men. there was no significant difference between the genders in the case of other subscales used to describe behavior or the subscales that assess the level of knowledge (table 1). seemedj 2019, vol 3, no. 1 information security and privacy awareness 24 southeastern european medical journal, 2019; 3(1) table 1. gender differences among healthcare employees *student’s t test in regard to qualifications, average estimation regarding personal computer maintenance was significantly higher for highly educated participants (one way anova, p = 0.01), while participants with a high school diploma were the group that was most aware of the importance of data protection and of the risk of loss of personal and professional data, money or identity on the internet (one way anova, p = 0.03), as seen in table 2. table 2. qualification differences among healthcare employees scales and subscales arithmetic mean (standard deviation) p* high school diploma (n = 176) bachelor’s degree (n = 69) master’s degree (n = 42) prb 4.01 (0.37) 4.02 (0.36) 4.09 (0.39) 0.52 ub 4.47 (0.55) 4.47 (0.49) 4.35 (0.42) 0.43 pcm 2.85 (0.87) 2.99 (0.84) 3.29 (0.88) 0.01† adl 4.72 (0.39) 4.62 (0.39) 4.61 (0.37) 0.08 ka 3.21 (0.54) 3.16 (0.48) 3.16 (0.41) 0.75 sc 3.29 (0.87) 3.39 (0.81) 3.18 (0.87) 0.46 sd 2.37 (0.90) 2.08 (0.70) 2.14 (0.75) 0.03‡ bq 3.97 (0.77) 4.00 (0.63) 4.16 (0.63) 0.29 *one way anova †between high school diploma and master’s degree (scheffé test) ‡between high school diploma and bachelor’s degree (scheffé test) a relatively low, statistically significant positive correlation was found between age and the “usual behavior” subscale, meaning that older participants display more secure online behavior on the internet (spearman’s correlation test, rho = 0.29, p < 0.001). likewise a very low, but statistically significant positive correlation was found between age and the “access data lending” subscale (spearman’s correlation test, rho = 0.13, p = 0.03), which mostly means that older participants lend their access data to other colleagues at work less often (table 3). scales and subscales arithmetic mean (standard deviation) p* male (n = 45) female (n = 242) prb 3.99 (0.42) 4.03 (0.36) 0.51 ub 4.29 (0.59) 4.48 (0.50) 0.03 pcm 3.10 (0.98) 2.92 (0.85) 0.22 adl 4.59 (0.43) 4.70 (0.38) 0.10 ka 3.10 (0.52) 3.20 (0.50) 0.20 sc 3.16 (0.98) 3.33 (0.83) 0.22 sd 2.12 (0.74) 2.30 (0.86) 0.18 bq 4.02 (0.80) 4.00 (0.70) 0.86 seemedj 2019, vol 3, no. 1 information security and privacy awareness 25 southeastern european medical journal, 2019; 3(1) table 3. age differences among healthcare employees scales and subscales age of healthcare employees (n = 287) rho 95% ci p* prb 0.09 -0.03 to 0.20 0.13 ub 0.29 0.18 to 0.39 < 0.001 pcm -0.08 -0.20 to 0.03 0.16 adl 0.13 0.01 to 0.24 0.03 ka 0.10 -0.01 to 0.21 0.09 sc 0.03 -0.08 to 0.15 0.58 sd 0.06 -0.05 to 0.18 0.30 bq 0.10 -0.01 to 0.22 0.09 *spearman’s correlation test average estimations per scale and subscale are shown in the first column of table 4. the lowest average estimation for participants was 2.27 ± 0.84 (x ± sd) for the “secured data” subscale, which measures awareness of privacy, while the highest average estimation 4.68 ± 0.39 (x ± sd) was for the “access data lending” subscale, which measures risky behavior (table 4). table 4. differences between healthcare employees and standardized behavioral norms among internet users in croatia scales and subscales arithmetic mean (standard deviation) p† healthcare employees (n = 287) standardized behavioral norms* (n = 4859) prb 4.03 (0.37) 4.00 (0.42) 0.24 ub 4.45 (0.52) 4.16 (0.59) < 0.001 pcm 2.95 (0.87) 3.27 (0.83) < 0.001 adl 4.68 (0.39) 4.66 (0.49) 0.50 ka 3.19 (0.51) 3.07 (0.53) < 0.001 sc 3.30 (0.85) 2.93 (0.82) < 0.001 sd 2.27 (0.84) 2.32 (0.87) 0.34 bq 4.00 (0.72) 3.83 (0.78) < 0.001 *average internet user in croatia (25) †student’s t test seemedj 2019, vol 3, no. 1 information security and privacy awareness 26 southeastern european medical journal, 2019; 3(1) the results of the comparison between the surveyed healthcare employees and the standardized behavioral norms among internet users in croatia collected in a national project (n = 4859) show statistically significant differences for the majority of the subscales, often in favor of healthcare employees (table 4). the surveyed healthcare employees achieved better results in the “usual behavior” subscale (student’s t test, p < 0.001), in the “security in communications” subscale (student’s t test, p < 0.001), and in the “backup quality” subscale (student’s t test, p < 0.001). they achieved better results in the overall “knowledge and awareness” scale (student’s t test, p < 0.001), but significantly worse results in the “personal computer maintenance” subscale (student’s t test, p < 0.001). discussion analysis of the results has shown that healthcare employees included in this study are statistically better than the average internet user in croatia when it comes to their knowledge about digital security and their potentially risky online behavior. however, their average estimations per scale and subscale are better than referent estimations only in part and are not very high, especially when it comes to awareness measured in the “security in communications” and “secured data” subscales. female users are the most skeptical of the surveyed healthcare employees, so they are more careful in their online behavior. the results were similar for older participants. both results are in accordance with previous studies based on the same questionnaire (11, 12). the results that show that participants with a high school degree are the most skeptical in regard to data protection and the risk of loss of personal and professional data, money or identity on the internet are also partially in accordance with those of previous studies. on the other hand, participants with higher education achieved better results when it comes to their personal computer maintenance. hospitals’ business information systems include both business and private data, i.e. digital data about healthcare processes and patients’ private information. since the user is often the weakest element of the information and communication system when it comes to data protection (1 4), healthcare employees have an obligation to be familiar with and behave in accordance with security protocols at their workplace. employees typically need additional education courses organized by their institution and alert messages sent on a regular basis by system administrators (14 17) in order to acquire better knowledge regarding security threats and to increase their own awareness regarding the importance of information security for the system, for the patients and for themselves (18, 19). some general recommendations for more secure behavior on the internet that may help healthcare employees and all other internet users are presented below (13): • limit posting personal data on the internet because what is once posted on the internet remains permanently recorded! • different systems are not equally secure or equally dangerous; you should exercise increased caution in an unknown setting! • user access data are personal and they are used to verify identity on the internet. they should be handled with extreme caution because they represent the electronic identity of a user. users should know that no one should ever request that he/she disclose their access data, neither a system administrator nor a bank clerk! • the internet is similar to the real world and public spaces. maintain a healthy dose of mistrust when communicating with strangers ¬– creating a fake identity on the internet is much simpler than in the real world! • keep your operating systems, applications you use, and particularly antivirus protection up to date, both on personal and portable devices, as well as on mobile phones. seemedj 2019, vol 3, no. 1 information security and privacy awareness 27 southeastern european medical journal, 2019; 3(1) • back up important documents and files periodically and copy them to another location, removed from the original data. • try to differentiate business communication from private communication. • a high-quality password significantly increases the level of security. use a combination of capital and small letters, numbers and special characters. some limitations of this study are the relatively small number of healthcare employees surveyed in only three hospitals. in addition, the participants were compared to standardized behavioral norms in croatia. however, those referent values do not refer to secure behavior and extensive knowledge of data protection, but to the average internet user. in a future research, a comparison is planned between healthcare employees and different target-groups, such as students, employees in the government sector or in the banking sector. as there exists a high risk of data loss because of the nature of business protocols, healthcare employees need more education and training in order for their awareness regarding the importance of information security and privacy to increase. acknowledgment (funding): this paper was financed by the croatian government office for cooperation with ngos and co-financed by the european union’s connecting europe facility, under the project named “safer internet centre croatia: making internet a good and safe place”, agreement number: inea/cef/ict/a2015/115320. the sole responsibility of this publication lies with the authors. the european union is not responsible for any use that may be made of the information contained therein. references 1. solic k, ilakovac v. security perception of a portable pc user (the difference between medical doctors and engineers): a pilot study. med glas (zenica) 2009; 6: 261 4. 2. sasse ma, brostoffand s, weirich d. transforming the 'weakest link' a human/ computer interaction approach to usable and effective security. bt technology journal 2001; 19: 122 31. 3. solic k, sebo d, jovic f, ilakovac v. possible decrease of spam in the email communication. proceedings of the 34th international convention on information and communication technology, electronics and microelectronics (mipro), opatija, croatia, 2011. p. 170 3. 4. thompson h. the human element of information security. ieee security & privacy 2013; 11: 32-5. 5. velki t, solic k, nenandic k. razvoj i validacija upitnika znanja i rizičnog ponašanja korisnika informacijskog sustava (uzpk). psihologijske teme 2015; 24: 401-24. 6. james fk, keith wr. computer networking: a top-down approach. 5. ed. addison wesley: university of massachusetts amherst, 2007. 7. varga m. zaštita elektroničkih podataka. tehnički glasnik 2011; 5: 61-73. 8. vukovic d. uvođenje integriranog bolničkog informacijskog sustava. medix 2004; 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p. 95 107 seemedj 2023, vol 7, no 1 sarcopenia in hemodialysis patients 42 southeastern european medical journal, 2023; 7(1) review article pathophysiological and diagnostic aspects of sarcopenia in hemodialysis patients 1 zvonimir sitaš 1*, dubravka mihaljević 1 1 department of nephrology, clinic for internal diseases, university hospital centre osijek; department of internal medicine and history of medicine, faculty of medicine osijek, j. j. strossmayer university, osijek, croatia *corresponding author: zvonimir sitaš, zvonimir.sitas@gmail.com received: feb 27, 2023; revised version accepted: mar 27, 2023; published: apr 30, 2023 keywords: sarcopenia, hemodialysis, chronic kidney disease, pathophysiology abstract chronic kidney disease and renal replacement therapy, particularly hemodialysis, contribute to the development of negative protein balance and muscle dysfunction in dialysis patients, from the development of protein-energy malnutrition to sarcopenia. due to multifactorial etiology and complex pathophysiological patterns, sarcopenia has proven to be a significant predictor of cardiovascular events and is associated with a higher risk of overall mortality. screening methods of chronic kidney patients and patients on hemodialysis who are at higher risk of developing sarcopenia, as well as diagnostic methods for this group of patients are not clearly defined, hence methods used for the general population of elderly patients, especially based on the revised european consensus on definition and diagnosis of sarcopenia of the european working group on sarcopenia in older people (ewgsop2), are utilized in this subpopulation as well. therefore, there is a need to define new biomarkers of sarcopenia such as the existing 24h urine excretion of creatinine, a product of estimated glomerular filtration of cystatin c and creatinine or myostatin and their use in routine work with dialysis patients to identify this condition among them and reduce morbidity and mortality. (sitaš z, mihaljević d. pathophysiological and diagnostic aspects of sarcopenia in hemodialysis patients. seemedj 2023; 7(1); 42-54) seemedj 2023, vol 7, no 1 sarcopenia in hemodialysis patients 43 southeastern european medical journal, 2023; 7(1) introduction the word sarcopenia comes from the greek σά ρξ (sarx) which translates to flesh and πενί α (penia) meaning loss. it was first introduced in 1989 by irwin rosenberg to describe the loss of muscle mass with aging leading to a reduced quality of life as a result of a higher risk of falls, fractures, hospitalizations and ultimately death (1). people suffering from this are at a higher risk of developing adverse outcomes due to bone and mineral disorder as a result of secondary hyperparathyroidism, then the higher rate of depression as a cause of development of anorexia and due to socioeconomic factors, affecting the quality of life that is significantly impaired in dialysis patients. it is estimated that the loss of muscle mass is 1–2% and muscle strength is 1.5–5% per year after the age of 50, primarily the musculature of the lower extremities (2), although muscle volume can be preserved due to myosteatosis and myofibrosis, which will be discussed below. in 1931, mr. critchley macdonald, a british neurologist, was the first to associate aging with a decrease in skeletal muscle mass (3). according to delmonico et al. in 1,678 subjects after the age of 70, the annual rate of muscle area reduction was 4.9±7.4% in men and 3.2±7.9% in women (4). the prevalence of sarcopenia varies widely in predialysis chronic renal patients of 6–14% (5, 6) and increases in patients on hemodialysis (hd) 4–64% (7, 8), without firm consensus on the prevalence of uremic sarcopenia, although some data suggest the prevalence of uremic myopathy of 50% (9). according to the metaanalysis of wathanavasin et al., the highest prevalence of sarcopenia is in europe (29.1%) and the lowest in the us (15.4%) (10). this metaanalysis also showed that sarcopenia is less diagnosed before the hemodialysis procedure (21.5%) compared to after hemodialysis (27.8%), primarily due to volume status, but without statistical significance. sarcopenia has also been shown to be one of the most important predictors of cardiovascular events (or 3.80 – 95% ci 1.79-8.09) with a high mortality risk (or 1.83 – 95% ci 1.40-2.39). low muscle strength and mass are independent factors of increased mortality in patients on hemodialysis (or 1.71 – 95% ci 1.20-2.44), all the more so, if they are associated (11). pathophysiology and etiology of sarcopenia knowledge of the physiology and histology of skeletal musculature is necessary for understanding the pathophysiology and histopathological changes within chronic kidney disease (ckd) and the uremic state. skeletal muscles belong to the group of transversely striped muscle tissue made of muscle fibers (actin, myosin), i.e. muscle cells that function as syncytium and are formed by fusion of myoblasts – precursor cells (12). skeletal muscle stem cells play a key role in muscle regeneration. various stimuli such as exercise, injury, stretching, etc. can activate these cells that are located between the plasma membrane and the basal lamina leading to asymmetric differentiation into myoblasts responsible for tissue regeneration and pluripotent stem cells responsible for self-renewal. myoblast differentiation requires primary myogenic regulatory factors myf5 and myod, as well as secondary – myogenin and myogenic regulatory factor 4 – mrf4 (13, 14). muscle fibers can be classified into three groups according to the isoform of the myosin heavy chain – slow oxidative red fibers (type i), fast oxidative red fibers (type iia) and fast glycolytic white fibers (type iix) that were represented in different proportions in skeletal muscles depending on muscle function (15). various factors are associated with histopathological changes in skeletal muscle in patients with advanced, i.e. terminal chronic kidney disease – especially a condition of chronic inflammation, which shows the replacement of muscle fibers with adipose tissue – myosteatosis, or fibrous tissue – myofibrosis and are a reflection of muscle dysfunction (16). in a study conducted on 60 patients on hemodialysis, the cross-section of muscle fibers was higher compared to the control group, which is explained by the development of interstitial edema in the interdialytic period, but the results also showed reduced oxidative capacity due to dysfunction seemedj 2023, vol 7, no 1 sarcopenia in hemodialysis patients 44 southeastern european medical journal, 2023; 7(1) of the enzyme succinate dehydrogenase and mitochondrial edema with a reduced network of capillaries (17). uremic toxins such as indoxyl sulfate, p-crescol and inorganic phosphorus, can impair myogenous differentiation in vitro and promote muscular atrophy in mice with ckd by promoting mitochondrial dysfunction (18, 19). development of sarcopenia and etiology of muscle dysfunction of patients on hemodialysis is multifactorial and arises from kidney disease itself, hemodialysis procedure and chronic inflammatory response, which contributes to reduced protein synthesis and increased degradation (20) (see figure 1). figure 1. etiological factors involved in the development of uremic sarcopenia the development of metabolic acidosis in ckd, vitamin d deficiency and insulin resistance also contribute to the negative protein balance. metabolic acidosis activates caspase-3 and the ubiquitin-proteasome system – two intracellular pathways responsible for protein degradation and promoting insulin resistance and growth hormone resistance (13, 21). 1,25(oh)2 vitamin d as an active form of vitamin d binds to vitamin d receptors in human muscle tissue, especially to c2c12 myoblasts, and causes reduced cell proliferation and promotes myogenic differentiation by myod expression and myostatin suppression (22). vitamin d deficiency also leads to reduced insulin secretion from pancreatic beta cells promoting insulin resistance (23, 7). insulin-like growth factor 1 (igf-1) shares a common intracellular pathway in protein synthesis with insulin, activating mtorc1 via pi3k (phosphatidylinositol-3kinase)/act leading to phosphorylation of foxo proteins and inhibiting their translocation into the nucleus. igf-1 suppresses both atrogin-1 and murf1 (ubiquitin ligase) which antagonizes skeletal muscle catabolism (24, 25). in patients on hemodialysis, the concentration of testosterone is reduced, which leads to the expression of myostatin and alteration of the igf-1 signaling pathway (26). myostatin is a protein that negatively affects the growth of muscle tissue through three signaling pathways. via activin, iib receptor activates smad2/smad3 and dephosphorylation act thus disrupting the igf-1 signaling pathway previously specified. it can also inhibit mtorc1 and induce apoptosis via the p38-caspase pathway (27). the hd procedure also contributes to the negative balance of protein metabolism. during the hd process, a significant loss of proteins and seemedj 2023, vol 7, no 1 sarcopenia in hemodialysis patients 45 southeastern european medical journal, 2023; 7(1) amino acids (> 5 g) was observed more pronounced at the beginning and end of the procedure itself (28). patients with ckd, especially patients on hd, have a decreased appetite due to the action of uremic toxins, chronic inflammation and hormonal disorders – primarily by a decrease in ghrelin and neuropeptide y, and an increase in leptin (29). chronic inflammatory condition of patients on hd is associated with the use of biocompatible membranes (30) and the role of the gastrointestinal tract or intestinal dysbiosis is increasingly mentioned. namely, due to dietary restrictions with reduced fiber intake, protein fermentation and exposure to endotoxins that induce the pro-inflammatory state and disrupt the gastrointestinal barrier increases (31, 32). proinflammatory cytokines such as il-6 and tnfalpha reduce the suppression of socs-3 protein through the activation of caspase-3 and the ubiquitin-proteasome system. il-6 also stimulates the expression of stat3 responsible for the expression of myostatin and leads to muscle degradation (33, 34). the production of pro-inflammatory mediators is induced by the formation of advanced glycation end products (age) that accumulate in patients with ckd due to reduced renal clearance and increased production (non-enzymatic changes in protein or lipid amino acids that are reduced by exposure to sugars or their metabolites) and lead to oxidative stress, insulin resistance and endothelial dysfunction (35, 36). yabuuchi et al. showed that the accumulation of age in the muscle tissue of mice leads to mitochondrial dysfunction (by reducing the activity of succinate dehydrogenase and pgc1-alpha and reducing the density of the capillary network) (37). furthermore, age can impair muscle function through associated membrane rage receptors whose activity reduces the expression of myogenin (38), impairs the distribution of muscle fibers and increases the rigidity of muscle connective tissue (39). these etiological factors contribute to negative protein balance that can eventually lead to the decline of muscle mass, muscle strength and lower physical activity because patients on hd are at a higher risk of developing sarcopenia compared to the general population and these changes occur at an earlier age. diagnosis of sarcopenia and associated conditions skeletal musculature is mostly made of protein and it is the best indicator of protein status. the development of protein-energy malnutrition (pem) – a term presented in 2007 by the international society of renal nutrition and metabolism (isrnm), occurs due to various pathophysiological mechanisms, that include nutritional and metabolic disorders in patients with chronic kidney disease and lead to the development of a state of chronic muscle and adipose tissue catabolism (40, 41). it is associated with chronic inflammation, uremia, anorexia due to decreased appetite, hypoalbuminemia, muscle loss with weight loss or no weight loss and poor clinical outcome. due to pem, there is a reduction in protein supplies and energy sources in patients with different ckd stages, leading to a decrease in the functional abilities of the patient. malnutrition, on the other hand, is an imbalance of energy intake, protein and other nutrients that leads to measurable undesirable effects on tissues and physical functions and can be presented with malnutrition, but also overfeeding (42–45), so we can find it in obese patients too. such a physiological state can lead to cachexia – a syndrome manifested by severe muscle loss with or without loss of fat tissue (46) and sarcopenia, which, according to the consensus of the ewgsop2 defines as „a progressive and generalized muscle disorder characterized by low muscle power and reduced quality and quantity of muscle mass“ (47). quantitative changes are related to the loss of muscle mass and volume, and qualitative to the loss of muscle strength and physical activity (48). in these revised guidelines, the emphasis is on muscle strength because it has a better predictive value for the occurrence of adverse outcomes. if the patient meets the criterion of low muscle strength, it is probably sarcopenia, i.e. presarkopenia, if it meets the second criterion of low muscle quality or quantity, the diagnosis can be confirmed by sarcopenia, and if it meets the third criterion of low physical activity, seemedj 2023, vol 7, no 1 sarcopenia in hemodialysis patients 46 southeastern european medical journal, 2023; 7(1) sarcopenia is considered severe. according to the international working group on sarcopenia (iwgs), sarcopenia is characterized by low skeletal muscle mass (with an increase in body fat) and decreased muscle function (49). ewgsop2 consensus provides a broader definition by dividing muscle strength from muscle activity and distinguishes severe sarcopenia. sarcopenia can be divided into primary (agerelated) when there is no other evident cause and secondary in systemic diseases especially those that can cause inflammatory processes including ckd, conditions of reduced physical activity (sedentary lifestyle, in bedridden patients) and some nutritional factors such as gastrointestinal diseases, malabsorption syndrome, insufficient food intake (9). the main difference between these two groups is that the primary group occurs over a continuous period and is more pronounced after the age of 50, while the secondary is more intense and depends on the conditions that cause it (50). unlike primary sarcopenia, sarcopenia in chronic kidney patients is characterized by more pronounced protein degradation. ewgsop2 subcategorizes sarcopenia into acute if it lasts less than 6 months and chronic if it lasts 6 months and more. such a division places emphasis on periodic assessments of sarcopenia in patients at increased risk (47). in elderly patients, a condition of sarcopenic obesity can be observed, which is presented by reduced muscle mass due to adiposity (51). for the diagnosis of sarcopenia, a multitude of different tests and methods can be used, the choice of which depends on the patient, the technical availability and the purpose of testing (whether we are examining the progression of the condition or recovery) – see figure 2. figure 2. sarcopenia diagnostic algorithm – adapted according to ewgsop2 level of strength, assistance with walking, rising from a chair, climbing stairs and falls are criteria in the 5-item questionnaire (sarc-f) as a screening method for patients with sarcopenia. it includes the patient’s subjective attitude about the ability to walk, get up from a chair, climb stairs, strength level, and the existence of falls in the past year. the questionnaire has high specificity in predicting low muscle strength and will detect more severe cases as such (52, 53). if the test is positive (sarc-f ≥4), the first criterion – muscle strength – is examined. to test muscle seemedj 2023, vol 7, no 1 sarcopenia in hemodialysis patients 47 southeastern european medical journal, 2023; 7(1) strength, the strength of the handshake can be measured with a jamar dynamometer, which is a simple and inexpensive method and correlates with the strength of other body regions, such as the strength of the upper and lower extremities (54, 55) and should be measured before the hemodialysis procedure (56, 57). the metaanalysis by hwang et al. showed that people with low muscle strength had a 1.88 times higher risk of overall mortality (58). vogt et al. suggested cut-off values that would suggest higher mortality in patients on hd, <7 kg in women and <22.5 kg in men (59), while according to ewgsop2, values <16 kg for women and <27 kg for men. in addition to testing the strength of the handshake, the test of getting up from the chair can be used, which predominantly examines the strength of the muscles of the lower extremities (quadriceps). the test measures the time it takes a patient to get out of the chair 5 times without arm support and the time > 15 s is considered significant. if these tests determine reduced muscle strength, it can be said that sarcopenia is probable and confirmed by testing muscle quality or quantity. muscle quantity can be shown as appendicular muscle mass (amm), total skeletal muscle mass (smm) and cross-section of a specific muscle group. computed tomography (ct) and magnetic resonance imaging (mri) are considered the gold standards in non-invasive assessment of muscle mass and myosteatosis (60, 61), but they are not widely used due to expensiveness, radiation exposure and necessary logistics. alternative methods include densitometry (dxa) and bioelectrical impedance analysis (bia) which can assess the total and appendicular muscle mass and in the case of bia and nutritional status of patients on hd with better evaluation of intracellular and extracellular fluid (62, 63) which should be done after the hd procedure. the absolute values of smm and amm can be modified according to weight (amm/weight), height square (amm/height2) or body mass index (amm/bmi). the cut-off values for these methods are according to ewgsop2 amm/height2 <7 kg/m2 for men and <5.5 kg/m2 for women. the method that can be done at the patient’s bedside is an ultrasound measurement of the thickness of the quadriceps femoris muscle (rectus femoris and vastus intermedius) which correlates with nutritional status (9). if any method determines low muscle quality or quantity, we can talk about the existence of sarcopenia whose weight is additionally assessed by the analysis of physical activity. most often, the walking speed test is used. the cut-off value is ≤ 0.8 m/s. the test is performed by measuring time at a distance of 4 m, walking at the usual walking speed (64, 65). diagnostic challenges of sarcopenia in ckd/dialysis patients although the diagnostic criteria of sarcopenia are well-defined in the general population, there is no consensus on the diagnosis of uremic sarcopenia in dialysis patients. most studies apply definitions relating to the elderly population leading to the heterogeneity of the results of the prevalence of sarcopenia in the dialysis population as previously stated. there is a risk of underestimating the prevalence of low muscle mass if in obese patients smm is modified according to the square of height and in such patients, there is a better modification according to body mass index (66). furthermore, several studies have shown that low muscle mass in dialysis patients is not associated with increased mortality unlike low muscle strength and low muscle activity defined by a weak handgrip test and slow gait speed (67, 68), and using this approach overlooks patients only with low muscle strength. this is supported by the kdoqi (kidney disease outcomes quality initiative) guidelines according to which hand grip strength is useful in assessing protein energy wasting because the use of bia and dexa is determined by volume status, so patients who are in overvolemia or with early changes in muscle structure would be misdiagnosed (69). fahal et al. (70) examined the contractile properties and muscular strength of dialysis patients with muscle quadriceps biopsy. the main difference between this group of patients and the control was poor muscle relaxation, which can affect muscle strength independently of muscle mass, which is supported by the fact seemedj 2023, vol 7, no 1 sarcopenia in hemodialysis patients 48 southeastern european medical journal, 2023; 7(1) that 45% of patients had type i and 40% of type ii muscle fibers atrophy and fibers type ii were significantly lower in malnourished patients, which again shows that muscle mass is not the only determinant of muscle strength. pereira et al. (71) define sarcopenia in ckd patients treated conservatively by reduced muscle function (hand grip strength – 30% percentile of the reference population), reduced muscle mass (measuring mid-arm muscle circumference) and reduced skeletal musculature index according to bioimpedance analysis (<6.76 kg/m2 in women and < 10.76 kg/m2 in men). furthermore, if low muscle mass is confirmed, patients may be in pronounced muscle wasting. therefore, in the assessment of sarcopenia in dialysis patients the focus should be on the components of low muscle strength and physical activity and such patients should be encouraged to change their lifestyle habits, diet and exercise for the preservation of muscle mass and muscle function. in addition to the aforementioned tests for diagnosis and monitoring of patients with sarcopenia, there was a need to define certain biomarkers for early detection of sarcopenia in patients with ckd. as an endogenous metabolite of skeletal muscle, the determination of creatinine excretion in 24h urine is justified in the assessment of muscle mass (72, 73) with a lack of adequate collection of all-day urine in certain patients. this problem was attempted to be solved by determining a new sarcopenia index – the product of the estimated glomerular filtration of cystatin c and creatinine (74). in the study of lin et al., this index was independently associated with muscle strength, mass and walking speed in patients with ckd (75). measurement of serum concentration of myostatin as a muscle consumption biomarker was also considered but with conflicting results of studies due to the influence of age, gender, inflammatory conditions, physical activity and metabolic syndrome. myostatin values are generally higher in patients with ckd, i.e. patients on hd with greater muscle strength and muscle mass compared to healthy individuals (27, 76). conclusions chronic kidney disease and renal replacement therapy, particularly hemodialysis, contribute to the multifactorial etiology of sarcopenia negative protein-energy balance and increased mortality of chronic kidney patients. adequate screening systems and diagnostic methods are crucial in the early recognition of patients at elevated risk. diagnostic tests and methods in the assessment of sarcopenia should be adapted to the conditions in which they are performed, patient groups and systematic monitoring. in conditions of unavailability of tests and methods adapted to dialysis patients, existing methods of assessing muscle strength, muscle quality and quantity, and physical activity can be used according to the ewgsop2 group of experts with a tendency to find new biomarkers of sarcopenia. due to the complex pathophysiology of protein catabolism, it is expected to find biomarker panels and their application in the stratification of heterogeneous groups of patients such as patients with ckd with the aim of adequate action and prevention of premature morbidity and mortality. the emphasis should be put on the fact that sarcopenia is a modifiable risk factor for adverse outcomes in ckd patients, using preventive nutritional measures based on data obtained by existing diagnostic methods and tests, so it should be further studied and adequately diagnosed and treated. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. seemedj 2023, vol 7, no 1 sarcopenia in hemodialysis patients 49 southeastern european medical journal, 2023; 7(1) references 1. rosenberg i. h. 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(2020). myostatin as a biomarker of muscle wasting and other pathologies-state of the art and knowledge gaps. nutrients, 12(8), 2401. https://doi.org/10.3390/nu12082401 author contribution. acquisition of data: zš, dm administrative, technical or logistic support: zš, dm analysis and interpretation of data: zš, dm conception and design: zš, dm critical revision of the article for important intellectual content: zš, dm drafting of the article: zš, dm final approval of the article: zš, dm guarantor of the study: zš, dm seemedj 2018, vol 2, no. 2 comorbidity of posttraumatic stress disorder 8 southeastern european medical journal, 2018; 2(2) original article comorbidity of posttraumatic stress disorder and alcohol dependence 1 tihana batrnek 1, mateo gašpert 1 1 faculty of medicine osijek, university of osijek, osijek, croatia corresponding author: tihana batrnek, tihana.batrnek@gmail.com received: mar 1, 2018; revised version accepted: mar 13, 2019; published: mar 31, 2019 keywords: alcoholism, comorbidity, ptsd, nursing care abstract aim: the aim of the research was to examine the extent to which alcohol dependence occurs in comorbidity with ptsd. methods: the study included 165 respondents hospitalized at the department of psychiatry in osijek. all respondents were previously diagnosed with ptsd. the overall questionnaire was designed specifically for this study, and it included information about age, gender, education, psychological problems, the continuum of treatment, and questions about alcohol consumption. results: most of the patients live with their wives and children: 34 (54%) of them from the 2004 study, and 40 (39.2%) of them from the 2014 study. also, 72.1% of the patients who responded to the questionnaire have family support. the marital status of the patients differed significantly over the years (χ² test; p = 0.007). results show that 35.2% consume alcohol daily. furthermore, there are significantly more patients who don`t consume alcohol with the prescribed therapy (χ² test; p = 0.042). conclusion: more than one third of the patients diagnosed with ptsd have alcohol dependence. the number of people diagnosed with ptsd and alcohol dependence increased in 2014 when compared to the 2004 findings, including the number of divorces and the consumption of alcohol with the prescribed therapy. (batrnek t, gašpert m. comorbidity of posttraumatic stress disorder and alcohol dependence seemedj 2018; 2(2); 8-16) seemedj 2018, vol 2, no. 2 comorbidity of posttraumatic stress disorder 9 southeastern european medical journal, 2018; 2(2) introduction several years after the croatian war of independence ended, croatia started to face its consequences, one of them being the rising number of war veterans, which totals up to around 27 000. a problem in particular are the disabled war veterans who suffer from posttraumatic stress disorder (ptsd). it is not just a private problem, but also a social one because veterans experience problems with the performance of daily work tasks, family problems, problems with relationships, and problems with everybody around them. in some cases, it is even possible to develop a dependence on alcohol or drugs (1). long-term effects of trauma are present within the psychological, social, and biological level. whether people will overcome their traumatic experiences or develop some of the pathological responses to trauma depends on the characteristics of their own trauma, personality characteristics, and the quality of social support (2). posttraumatic stress disorder posttraumatic stress disorder is defined as a pathological anxiety that usually develops after an individual experiences or witnesses severe trauma. initially, a person responds with intense fear, helplessness and horror. later, the disorder develops a response to an event marked by persistent reenactment of events and symptoms of emotional numbness, avoidance and hyperarousal. ptsd can be acute, chronic and delayed (3). war itself contains probably the largest number of some of the most powerful provoking factors. when people fight in war, they must give up on their moral standards which are necessary to sustain civilized life (4). the symptoms and the fully developed clinical picture of ptsd can occur immediately after a stressful event or shortly thereafter, but the initial symptoms and psychological problems may occur many years after the trauma had occurred. a person usually develops anxiety and depression immediately after experiencing a trauma. the most important characteristic of ptsd is constantly re-experiencing the traumatic event. the patient usually faces incentives from the environment, as well as some agonizing and unwanted memories of the traumatic event. these intrusive memories are accompanied by a corresponding painful, emotional reaction. the patient may suddenly begin to behave or feel as if the traumatic eventis re-occuring. they try to avoid thoughts or feelings associated with the trauma, or the activities and situations which can remind them of the trauma. ptsd can cause sleep disturbances, excessive tension, hypersensitivity, and some problems with concentration and reactions marked by excessive fear. also, ptsd can cause aggressive reactions of variable intensity. the treatment of ptsd is a combination of drug treatment and psychotherapy. when a traumatic event is weaker, the intensity and the psychological reactions are less expressed, hence there is no need for therapeutic intervention, except for social support. in situations where the same person is experiencing several consecutive traumatic events, they require support from their families and friends, and only in certain situations an expert-centered aid. group psychotherapy plays a central role in the psychiatric treatment of patients with ptsd (5). the aim of psychotherapy is to reduce the severity of symptoms and to gain more mature adaptive mechanisms. this provides better integration and reintegration into the family, as well as the social environment. during such group sessions, therapists work hard to establish trust, safety, and respect, and they encourage psychoeducation, altruism, acceptance, exposure of a traumatic experience and interpersonal learning (6). alcohol dependence alcoholism is a progressive, fatal disease characterized by a loss of control of drinking alcoholic beverages, an obsession with alcohol, and a denial of the link between alcohol consumption and deterioration of health and life chances (7, 8). alcoholism can also be defined as a state with active signs and symptoms of excessive drinking of alcoholic beverages, including increased tolerance to alcohol and seemedj 2018, vol 2, no. 2 comorbidity of posttraumatic stress disorder 10 southeastern european medical journal, 2018; 2(2) behavioral changes (9). alcohol causes reduction of concentration, vision, the ability to differentiate between the light stimuli, as well as alertness and attention. alcoholics are used to drinking alcohol in order to cover up their negative feelings of anger, guilt and depression. the consequences of a long-term alcohol consumption are manifested through fatal damagings to many organ systems, as well as mental disorders. gradually, alcohol dominates their thoughts, feelings and actions (10). the diagnosis of alcohol dependence can only be set if three or more of the following criteria are observed during the previous year: a strong desire or urge to drink, difficulties in self-control if a person tries to quit drinking, a physiological state of abstinence, an evidence of tolerance, such as increasing the amount of alcohol required to achieve effects which were previously caused by a small amount, a progressive negligence of alternative pleasures, or interests caused by drinking and continuation of alcohol consumption despite clear evidence of its adverse effects. the goal of the treatment is to establish and maintain permanent abstinence from alcohol, as well as to change the lifestyle and the value systems which led to excessive alcohol consumption in the first place. the treatment should include the patient’s family, because alcoholism is often a family problem (3). comorbidity of posttraumatic stress disorder and alcohol dependence additional diagnoses may affect the patients’ treatment during their stay in the hospital, namely in terms of the need for diagnostic tests, nursing care, and monitoring. additional diagnoses may also affect the already provided nursing care and cause a significant increase in costs and a longer stay in the hospital (11). posttraumatic stress disorder can occur in comorbidity with other mental and/or physical disorders. comorbidity occurs in 80% of the cases (12). ptsd often co-occurs with alcoholism, depressive disorders, addictions to psychoactive substances, and personality disorders (13). there wasn`t much therapy and rituals for the majority of croatian veterans as for the veterans of other wars. the lack of social care and support, especially the latter notion, left many croatian war veterans unexperienced in terms of returning and, in actuality, unable to ever fully return from the war (4). a nurse working with ptsd patients has an important role in the preservation of their personal and social integrity. the most important interventions are psychosocial support, education, and counseling. education and psychosocial support help the patient and his/her family to deal with the disease and to change the patient’s lifestyle, namely in a way to cope with the changes in the pattern of daily activities, which ultimately contributes to improving the quality of life. although the development of ptsd depends on many factors, the education of nurses plays an important role both in its treatment and prevention. through education we raise the level of knowledge of traumatized people about the disorder itself, its symptoms, and the subsequent behavior. nurses provide support to patients and they encourage patients to talk with their families and friends about traumatic experiences (14). the aim of the research of comorbidity of posttraumatic stress disorder and alcohol dependence was to examine the extent to which alcohol dependence occurs in comorbidity with ptsd. objective and methods the study was structured retrospectively, namely on the basis of the criteria for inclusion of the ptsd-diagnosed subjects listed in the medical documentation. ptsd was previously diagnosed by a psychiatrist. the study included 165 patients hospitalized at the department of psychiatry in osijek. 63 patients were hospitalized in 2004, whereas 102 were hospitalized in 2014. in the medical documentation of the subjects diagnosed with ptsd, all comorbidity was sought and the focus was on the diagnosis of alcoholism. the general form was designed for the purpose of this research, and it included information about age, gender, education, job and marital seemedj 2018, vol 2, no. 2 comorbidity of posttraumatic stress disorder 11 southeastern european medical journal, 2018; 2(2) status, family structure, the existence of physical illness, some lasting psychological problems, the continuity of treatment, and questions about alcohol consumption and the war. ethical permission for the study was sought and provided by the clinical hospital centre in osijek. the research was conducted in an ethical and responsible manner, and is in full compliance with all relevant codes of experimentation and legislation. statistical methods to describe the frequency distribution of the variables, descriptive statistical methods were used. all variables were tested to normality distribution with the kolmogorov-smirnov test. the middle values of continuous variables were expressed as an arithmetic mean/average and standard deviation for normally distributed variables, as well as the median and the range of variables which are not normally distributed. to determine the difference between two independent samples, the t-test was used, and for more than two samples, the anova kruskal wallis test was used. to determine the difference between the proportions, the χ2 test was used. all p values were two-sided. the level of significance was set at α = 0.05. for the statistical analysis, the statistical program spss (version 16.0, spss inc., chicago, il, usa) was used. results the average age of the patients examined was 41.67 years in 2004, and 51.16 in 2014. in 2004, was 63 patients, and 29 of them (46%) belonged to the 29-39 age group. in 2014, was 102 patients, and 54 (52.9%) were in the age group of 51 and over. all patients in 2004 were male. in 2014, 98 patients (96.1%) were male, and only 4 (3.9%) were female. most of the patients have completed high school education. out of a total of 165 patients, 129 (78.2%) had high school education. in 2004, 17 (27%) patients were unemployed, 16 (25.4%) were employed, and 30 (47.6%) were retired. during 2014, 23 (22.5%) patients were unemployed, 16 (15.7%) were employed, and 63 (61.8%) were retired. the marital status of the patients examined varied significantly over the years (χ² test; p = 0,007). the collected data are presented in table 1. table 1. marital status of patients marital status total married single divorced widow cohabitation year 2004 quantity 52 6 3 0 2 63 % 82,5 % 9,5 % 4,8 % 0,0 % 3,2 % 100,0% 2014 quantity 59 15 23 3 2 102 % 57,8 % 14,7 % 22,5 % 2,9 % 2,0 % 100,0% total quantity 111 21 26 3 4 165 % 67,3 % 12,7 % 15,8 % 1,8 % 2,4 % 100,0% the study shows that there is 14.5% of the patients without children, whereas 20.6% of the patients have one child. patients with more than one child relate to the following numbers: 41.2% (two children), 17.3% (three children), 3.6 % (four seemedj 2018, vol 2, no. 2 comorbidity of posttraumatic stress disorder 12 southeastern european medical journal, 2018; 2(2) children), 1.2% (five children), and 1.2% (six children). most of the patients live with their wives and children: 34 (54%) of them from the 2004 study, and 40 (39.2%) of them from 2014 study. the number of the patients who live with their parents increased: only 2 (3.2%) patients lived with their parents in 2004, while in 2014, 14 (13.7%) lived with their parents. the number of patients who live alone also increased. during 2004, only 9 (14.3%) patients lived alone, while during 2014, 26 (25.5%) lived alone (χ² test; p = 0,032). results have shown that 72.1% of the patients examined have family support. the largest number of patients, 130 (78.8%), lived in their own houses or apartments. in 2004, 2 (3.2%) patients lived with their parents, while in 2014 the number increased to 16 (15.7%) (χ² test; p = 0,010). also, in 2014, 16 patients lived with parents (15.7%). during 2004, 2 (3.2%) patients were tenants, while in 2014 there were 12 (11.8%) of them living as tenants. during 2004, 33 (52.4%) patients lived in the city, whereas in 2014, 53 (52%) more patients lived in the countryside. overall, out of 165 patients, 83 (50.3%) lived in the countryside. if we look at the patients' answers to the question of age at the beginning of the war, the arithmetic mean approximated to 28.27 years; the minimum value was 16 years, while the maximum one was 55 years. if we look at the patients' period of involvement during the war, they participated actively for approximately 3.61 years. the minimum value of the war service was less than a year, while the maximum value was 6 years. out of 165 patients in 2004 and 2014, 124 (75.2%) have not suffered from injuries during the war. also, 158 (95.8%) have not experienced the loss of a close person during the war. out of 165 patients, 132 (80%) had no problems with the law. within the remaining 20% of the patients, the majority (14 of 33) consumed alcohol daily. the data regarding the distribution of patients, considering their alcohol consumption, are presented in table 2. the majority of the patients (159 = 96.4%) do not consume alcohol with drugs. during 2004, 5 (7.9%) patients consumed alcohol with the prescribed therapy, whereas in 2014, 20 (19.6%) patients consumed alcohol with the prescribed therapy. results show that there are significantly more patients who don`t consume alcohol with the prescribed therapy (χ² test; p = 0,042). table 2. distribution of respondents considering alcohol consumption alcohol consumption total every day several times a week several times a month do not consume alcohol year 2004 quantity 15 0 0 48 63 % 23,8% 0,0% 0,0% 76,2% 100,0% 2014 quantity 43 3 7 49 102 % 42,2% 2,9% 6,9% 48,0% 100,0% total quantity 58 3 7 97 165 % 35,2% 1,8% 4,2% 58,8% 100,0% none of the patients from the 2004 study were psychiatrically treated before the war. the 2014 study, however, shows that 3 (2.9%) patients were psychiatrically treated before the war. seemedj 2018, vol 2, no. 2 comorbidity of posttraumatic stress disorder 13 southeastern european medical journal, 2018; 2(2) in the 2004 study, 16 (25.4%) patients were shown to have had some medical problems (χ² test; p = 0,011), whereas the number increased to 46 (45.1%) in the 2014 study. during 2004, diabetes mellitus, epilepsy and hypertension were the most common medical problems. during 2014, 20 (43.3%) patients had hypertension, whereas 9 (19.6%) suffered from diabetes mellitus and gastritis. during 2004, 15 (23.8%) patients have been diagnosed with alcohol dependence cooccurring with ptsd, and in 2014 there were 43 (42.2%) patients. there were other co-morbidities which are presented in table 3. table 3. comorbidities year disorder 2004 2014 acute stress reaction 0 4 anxiety depressive disorder 3 3 bipolar affective disorder 0 2 depression 1 0 depressive episode 3 2 depressive disorder 9 0 antisocial personality disorder 1 0 emotionally unstable personality 0 2 gambling 0 1 crisis situation 1 6 intentional self-harm 0 1 chronic depressive disorder 1 1 alcohol addiction 15 43 delirium 1 0 the emotional unstable personality structure 1 0 panic disorder 1 0 persistent delusional disorder 0 2 personality disorder 1 14 adjustment disorder 1 3 returning depressive disorder 4 32 psychosis paranoides 0 1 reactio psyhotica 0 2 psychosis 4 0 schizophrenia 1 0 delusional disorder 1 0 discussion this research compared the medical documentation of the patients diagnosed with ptsd on the basis of the studies conducted in 2004 and 2014. some of the results were expected. almost half of the patients in 2004 were between 29 and 39 years, while in 2014, most of patients were over 51, from which we can conclude that the population is aging. the seemedj 2018, vol 2, no. 2 comorbidity of posttraumatic stress disorder 14 southeastern european medical journal, 2018; 2(2) majority of the patients examined were married. the reassuring fact is that they also have family support, which is very important for posttraumatic stress disorder. the results associated with this fact relate to the fact that more than half of the patients live with their spouses and children. during hospitalization, the inclusion is very important, particularly the inclusion of family in the therapeutic treatment. families can ease the situation and motivate the patients to make progress. in all situations, nurses must support the patient's active role and prepare him/her for independent living (15). a surprising result, however, is the increased number of divorced patients in 2014. there were 23 divorced patients compared to 2004 when there were only 3 divorced patients. in 2004, all 3 divorced patients had been diagnosed with alcoholism. in 2014 there were 23 divorced patients, 14 of which had been diagnosed with alcoholism. all divorced patients who have been diagnosed with alcoholism consumed alcohol daily. such people are usually, but not necessarily, on their own, and it is harder for them to cope with the situation. problematic drinking is one of the potential and negative health outcomes of the divorce (16). marital dissolution can lead not only to increases in the quantity of the alcohol consumed (17), but also to increases in drinking-related problems (18). possible explanations for this phenomenon involve mediation by divorce-related stress, decreases in family obligations, and changes in the social network and socializing behaviors (19). however, as with other mental health outcomes, individuals are likely to vary in the way the divorce affects their drinking behavior. it is obvious that alcoholism has a greater impact on marital status than ptsd. in this study, most of the patients had no problems with the law, but the majority of those who do also suffer from alcohol dependence. this confirms the results of the previous studies which have shown that among the evaluated offenders, 57% was associated with addiction to alcohol and/or drugs, as opposed to 43% of the crimes associated with other mental disorders (20). the aim of one of the research was to analyze the impact of alcoholism in expressing and controlling aggression in war veterans with chronic ptsd. the studied sample included 240 war veterans with chronic ptsd. the results show that deprivation, aggression (p <0.001) and opposition (p <0.05) were more expressed in the respondents diagnosed with ptsd and alcoholism, as opposed to those whose ptsd diagnosis was not associated with alcoholism. in the patients who have been diagnosed both with ptsd and alcoholism, there is a statistically significant and predominant aggression apparent on all subscales, especially in comparison to subjects who have been diagnosed only with ptsd (21). this study shows that more than one third of the patients diagnosed with ptsd suffer from alcohol dependence. these findings are related to the majority of negative results connoted with divorce, problems with law, etc. in 1999, there was research on co-morbidity of ptsd and alcohol dependence which concluded that 52.2% of the patients examined were taking medium and large amounts of alcohol during the war. ptsd is often associated with chronic alcoholism for the purpose of self-healing (22, 23). in general, men drink more often than women. however, habits, and the intensity of alcohol drinking differ throughout the world and are influenced by a number of factors, such as gender, age, and various social and economic factors. in croatia, 81.3% of the male population and 51.2% of the female population drink frequently. alcohol addiction is slightly more common in middle aged people who experience lower incomes and who are less educated. as for the level of education, alcoholism is more common in people with higher education, situated in an urban environment, whereas in rural areas alcoholism is predominantly associated with a lower education level (10). taking all of the previously mentioned findings into consideration, the fact that this study has shown an increased number of alcoholconsuming patients with the prescribed therapy poses a serious issue. such outcomes can have many negative effects on health, and in no way can contribute to improving mental health. seemedj 2018, vol 2, no. 2 comorbidity of posttraumatic stress disorder 15 southeastern european medical journal, 2018; 2(2) conclusion based on the study and the results, we can conclude that most of the patients diagnosed with ptsd have family support. more than one third of the patients diagnosed with ptsd suffer from alcohol dependence. the number of people diagnosed with ptsd and alcohol dependence increased in 2014 when compared to the 2004 study, as well as the number of patients who consume alcohol with the prescribed therapy. the situation is the same with the number of the divorced patients in 2014 when compared to the 2004 study, and most of the divorced patients suffer from alcohol dependence. whereas most of the patients have no trouble with the law, the majority of those who do also suffer from alcohol dependence have troubles with the law. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare references 1. plačko v. vojni invalidi domovinskog rata na području križevaca i posttraumatski stresni poremećaj. polemos. 1999; 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41: 164-173. gruden v, gruden v jr, gruden z. ptsd and alcoholism. coll.antropol. 1999; 2: 607 610. seemedj 2018, vol 2, no. 2 stress and stressors for surgical nurses 48 southeastern european medical journal, 2018; 2(2) original article work-related stress and most common stressors for surgical nurses 1 stana pačarić 1,2,3,, ana nemčić 2, nikolina farčić 1,2,3 1 faculty of medicine osijek, josip juraj strossmayer university of osijek, osijek, croatia 2 university hospital center osijek, department of surgery, osijek, croatia 3 nursing institute "professor radivoje radić", faculty of dental medicine and health, osijek, josip juraj strossmayer university of osijek, osijek, croatia corresponding author: nikolina farčić, nikolina.farcic@mefos.hr received: dec 12, 2017; revised version accepted: mar 26, 2019; published: mar 31, 2019 keywords: occupational stress, general surgery, nurses abstract aim: the objective of this research was to determine the stress level in nurses at the department of surgery, clinical hospital osijek, and to identify the most common stressors. methods: the research included 105 nurses, 29 (28%) were men and 76 (72%) of them were women. it was conducted anonymously, by standardized questionnaire occupational stress questionnaire for hospital health care workers. results: the total scale of stress was 3.2 (interquartile range from 2.6 to 3.7) with no significant differences by gender. the results showed no statistically significant differences in the level of stress in terms of age, total length of service and level of education. the most common stressors in surgical nurses are insufficient number of employees, work overload, administrative work, a 24-hour responsibility, inappropriate public criticism, fear of infection, conflict with superiors, night and overtime work and pressure of set deadlines. conclusion: the greatest stress in surgical nurses, was connected with the work organization and financial issues, and in a group of women, a large share of stress was connected with the public criticism and litigation. (pačarić s, nemčić a, farčić n. work-related stress and most common stressors for surgical nurses. seemedj 2018; 2(2); 48-58) seemedj 2018, vol 2, no. 2 stress and stressors for surgical nurses 49 southeastern european medical journal, 2018; 2(2) introduction stress at work has been recognised worldwide as the greatest challenge for workers’ health and for their organisations (1). mcgrath (1973) defines stress as a significant imbalance between the demands and the ability to meet them, in a situation where a failure to satisfy the requirements has, according to the workers’ estimations, significant consequences (2). stress at work is a specific type of stress originating in the work environment. medical profession is classified as a high-stress profession due to a high level of responsibility for human lives and health, exposure to specific stressors, such as chemical, biological and physical hazards, and shift work (3). unlike the majority of other people, nurses face real suffering, pain and death on a daily basis. nursing is, by its nature, an extremely stressful profession (4). members of surgical teams are exposed to common stressors, such as overtime work, shift work and extremely demanding requirements (5). at the same time, good leadership, close cooperation and teamwork, as the essential qualities of the members of a surgical team, can impose additional work-related requirements, consequently accumulating more work-related stress (6). work in the operating room is one of the most exciting and demanding activities performed by the nurse, but it is also one of the most stressful health care jobs. teamwork is the key element of working in the operating room and it has been increasingly recognised that acute stress has a harmful effect on teamwork and communication between team members (7). operating nurses should have a high level of psychophysical stability and use constructive stress-response strategies, as those are considered the essential qualities of nurses working in the operating room (8). development of medicine and technology has increased the scope of work performed by nurses working in the operating room. as efficiency and promptness are the most important requirements and the staff is constantly under stress, the or department should therefore increase the amount and quality of stress relief courses (8). nurses working in intensive care units are exposed to a higher level of stress than those working in other departments (9). a research conducted in intensive care units in brazil examined the presence of work-related stress and dissatisfaction with activities that are considered critical for intensive care units. symptoms related to cardiovascular, digestive and musculoskeletal disorder, present in nurses working in intensive care units, are closely connected with the characteristics of particular health areas, which causes dissatisfaction and stress symptoms (10). burnout syndrome is a state of mental, physical or mental and physical exhaustion caused by excessive and prolonged stress (11). a study among nurses has shown that higher levels of burnout caused by work-related stress are associated with lower job satisfaction (12). surgical nurses have indicated different factors of job satisfaction (13) and stress (9) in comparison with other nurses and they also expressed higher motivation (14). the objective of the study is to determine (i) the stress level and (ii) the most common stressors for nurses at the department for surgery of the clinical hospital osijek and (iii) to determine the differences in the impact of stress among nurses in relation to their gender, age, education and length of service. subjects and methods a cross-sectional survey among health care workers, operating nurses employed at the department of surgery in the intensive care units and operating rooms at the clinical hospital osijek was conducted in may and june 2015. research included 105 participants, of which 29 (28%) were men and 76 (72%) were women. the majority of participants, 79 (76.7%) of them, had secondary education qualifications. the mean age of participants was 38 years (the interquartile range of 30-48 years) and the mean seemedj 2018, vol 2, no. 2 stress and stressors for surgical nurses 50 southeastern european medical journal, 2018; 2(2) length of service was 16.5 years (the interquartile range of 8-26 years), with no significant differences by gender. the participants were informed about the research in written and oral form. before the survey was conducted, the participants had read the instructions as an introduction to the main objective of the research. in the instructions, it was also emphasised that participation in the research was anonymous and voluntary. afterwards, the respondents gave their written consent for participation in the study. it took fifteen to twenty minutes for the participants to complete the questionnaires. completed questionnaires were submitted to the examiner personally in an envelope in order to ensure anonymity of the research. the ethics committee of the clinical hospital osijek gave its consent to conduct the research. the standardised occupational stress questionnaire for hospital health care workers developed by milan milošević (2009), which consists of 37 workplace stressors, was used in this research (15). based on a preliminary research, it was estimated that some issues (fear of exposure to cytostatics, constant responsibilities during 24-hour shifts and time limits for patient examination) were not particularly relevant for the study because the participants do not work with cytostatics, do not work in 24-hour shifts and do not perform patient examinations. the first part of the questionnaire included 34 workplace stressors classified under six factors. the stressors were related to work organisation and financial issues, public criticism and litigation, occupational risks and hazards, conflicts and poor communication, shift work and professional requirements. the participants estimated the stressors on the likert scale by rating them from 1 to 5, where 1 was (not stressful at all), 2 (rarely stressful), 3 (occasionally stressful), 4 (stressful) and 5 (extremely stressful). the second part of the questionnaire consisted of general data relating to sociodemographic characteristics (gender, age, education and total length of service). statistical methods figures were described by a median and interquartile range. categorical variables were described in absolute and relative frequencies. fisher's exact test and the mann-whitney test were used to examine the differences between independent groups (by age, gender and education). the level of significance was set at α = 0.05. data analysis was done by the spss software system for windows (version 13.0). results the study included 105 participants, of which 76 (72%) were women. table 1 presents the comprehensive data about the participants’ age, length of service and education qualifications categorised by gender (table 1). seemedj 2018, vol 2, no. 2 stress and stressors for surgical nurses 51 southeastern european medical journal, 2018; 2(2) table 1. participants by characteristics and gender men women total p total 29 (28%) 76 (72%) 105 (100%) age* 40 (28–49.5) 38 (31.5–47) 38 (30–48) 0.985† length of service* 19 (7.5–28) 16 (8.5–25) 16.5 (8–26) 0.894† qualifications secondary school qualifications 24 (82.8%) 55 (74.3%) 79 (76.7%) 0.770‡ college qualifications 4 (13.8%) 15 (20.3%) 19 (18.4%) university qualifications 1 (3.4%) 4 (5.4%) 5 (4.9%) age groups up to 30 years 10 (34.5) 16 (21.1) 26 (24.8) 0.348‡ 31 – 40 6 (20.7) 27 (35.5) 33 (31.4) 41 – 50 8 (20.7) 23 (30.3) 31 (29.5) 51 and over 5 (17.2) 10 (13.2) 15 (14.3) length of service groups up to 10 years 11 (37.9) 20 (26.3) 31 (29.5) 0.286‡ 11 – 20 5 (17.2) 26 (34.2) 31 (29.5) 21 – 30 8 (27.6) 22 (28.9) 30 (28.6) 31 and over 5 (17.2) 8 (10.5) 13 (12.4) * median (interquartile range); † mann-whitney test; ‡ χ2 test the majority of participants stated that stress related to work organisation and financial issues is caused mostly by an insufficient number of workers (49 participants, i.e. 46.7%) and work overload (40 participants, i.e. 38.8%). besides that, 23 participants (21.9%) rated public criticism as extremely stressful (table 2). table 2. participants by level of stress related to work organisation and financial issues and to the public criticism and litigation number (%) of participants not stressful at all rarely stressful occasionally stressful stressful extremely stressful total work organisation and financial issues inadequate income 3(2.9) 14(13.5) 29(27.9) 39(37.5) 19(18.3) 105(100) inadequate financial resources 2(1.9) 10(9.5) 30(28.6) 46(43.8) 17(16.2) 105(100) inadequate workspace 5(4.8) 25(23.8) 33(31.4) 32(30.5) 10(9.5) 104(100) little opportunity for promotion 9(8.7) 22(21.2) 42(40.4) 18(17.3) 13(12.5) 105(100) poor communication with superiors 8(7.6) 33(31.4) 33(31.4) 18(17.1) 13(12.4) 105(100) insufficient number of workers 3(2.9) 7(6.7) 17(16.2) 29(27.6) 49(46.7) 105(100) poor work organisation 8(7.7) 21(20.2) 25(24) 27(26) 23(22.1) 103(100) unexpected situations on a daily basis 4(3.8) 12(11.4) 35(33.3) 34(32.4) 20(19) 103(100) administrative work 5(4.8) 3(2.9) 29(27.6) 29(27.6) 39(37.1) 105(100) work overload 4(3.9) 5(4.9) 12(11.7) 42(40.8) 40(38.8) 105(100) public criticism and litigation threat of lawsuits 12(11.4) 16(15.2) 27(25.7) 29(27.6) 21(20) 105(100) inadequate expectations of patients 7(6.7) 13(12.4) 40(38.1) 29(27.6) 16(15.2) 105(100) inappropriate public criticism 4(3.8) 16(15.2) 29(27.6) 33(31.4) 23(21.9) 104(100) misinforming patients 5(4.8) 19(18.1) 47(44.8) 22(21) 12(11.4) 105(100) conflicts with patients 6(5.7) 27(25.7) 34(32.4) 25(23.8) 13(12.4) 105(100) non-separation of professional from private life 15(14.4) 20(19.2) 45(43.3) 16(15.4) 8(7.7) 105(100) 24-hour shifts 11(10.6) 13(12.5) 18(17.3) 37(35.6) 25(24) 103(100) seemedj 2018, vol 2, no. 2 stress and stressors for surgical nurses 52 southeastern european medical journal, 2018; 2(2) conflict with superiors was rated as an extremely stressful event by 21 participants (20%). an equal number of the participants rated night work as either extremely stressful (24.8%) or not stressful at all (20%) (table 3). considering professional requirements, the pressure arising from imposed time limits represented the most significant stressor for 20 participants (19%), whereas 15 participants (14.3%) rated the introduction of new technology as not stressful at all (table 3). table 3. participants by level of stress related to occupational risks and hazards, to conflict and poor communication and to shift work and professional requirements total rating on the scale of stress was 3.2 (interquartile range from 2.6 to 3.7) and there were no significant differences by gender. the most significant stressors were related mostly to work organisation and financial issues, whereas female respondents stated that a considerable amount of stress arises from public criticism and litigation (mann-whitney test, p = 0.046) (table 4). number (%) of participants not stressful at all rarely stressful occasionally stressful stressful extremely stressful total occupational risks and hazards fear of ionizing radiation 17(16.2) 35(33.3) 22(21) 22(21) 9(8.6) 105(100) fear of inhalation anaesthetics 29(28.2) 33(32) 24(23.3) 10(9.7) 7(6.8) 105(100) fear of infection 13(12.4) 29(27.6) 24(22.9) 28(26.7) 11(10.5) 104(100) fear of injury with a sharp object 13(12.5) 32(30.8) 25(24) 19(18.3) 15(14.4) 105(100) facing incurable patients 7(6.7) 17(16.2) 37(35.2) 26(24.8) 18(17.1) 105(100) conflicts and poor communication conflicts with colleagues 9(8.6) 23(21.9) 35(33.3) 25(23.8) 13(12.4) 105(100) conflicts with other associates 10(9.5) 21(20) 41(39) 22(21) 11(10.5) 105(100) poor communication with colleagues 14(13.3) 20(19) 39(37.1) 22(21) 10(9.5) 104(100) conflicts with superiors 15(14.3) 20(19) 26(24.8) 23(21.9) 21(20) 105(100) shift work night work 21(20) 18(17.1) 18(17.1) 22(21) 26(24.8) 105(100) shift work 14(13.5) 26(25) 22(21.2) 24(23.1) 18(17.3) 105(100) overtime 16(15.2) 20(19) 21(20) 24(22.9) 24(22.9) 104(100) professional requirements introduction of new technologies 15(14.3) 22(21) 40(38.1) 12(11.4) 16(15.2) 105(100) overload of information from profession 11(10.5) 21(20) 45(42.9) 12(11.4) 16(15.2) 105(100) lack of appropriate permanent education 13(12.4) 20(19) 44(41.9) 19(18.1) 9(8.6) 104(100) unavailability of literature 12(11.4) 25(23.8) 46(43.8) 10(9.5) 12(11.4) 105(100) pressure of imposed time limits 6(5.7) 18(17.1) 25(23.8) 36(34.3) 20(19) 105(100) seemedj 2018, vol 2, no. 2 stress and stressors for surgical nurses 53 southeastern european medical journal, 2018; 2(2) table 4. average rating on stress scale by gender median (25% 75%) by gender p* men women total work organisation and financial issues 3.5 (2.8-3.8) 3.5 (3.1-4) 3.5 (3.1-4) 0.137 public criticism and litigation 2.9 (2.4-3.7) 3.4 (2.8-3.9) 3.4 (2.6-3.9) 0.046 occupational risks and hazards 2.4 (1.8-3.7) 2.9 (2.3-3.6) 2.8 (2-3.6) 0.352 conflicts and poor communication 3 (2.3-3.8) 3 (2-4) 3 (2-3.8) 0.829 shift work 3 (2-4) 3 (2-4) 3 (2-4) 0.738 professional requirements 3 (2.3-3.4) 3 (2.4-3.6) 3 (2.4-3.6) 0.291 total rating 3 (2.5 3.6) 3.3 (2.7-3.8) 3.2 (2.6-3.7) 0.167 * mann-whitney test occupational risks and hazards were the sources of stress for participants aged between 31 and 40. although some variations were observed, they were not particularly significant (table 5). table 5: average rating on stress scale by age median (25% 75%) by age groups p* up to 30 years 31 – 40 41 50 51 and over total work organisation and financial issues 3.3 (3-3.7) 3.6 (3.1-3.9) 3.5 (3.1-4) 3.9 (3.5-4.2) 3.5 (3.1-4) 0.111 public criticism and litigation 2.9 (2.6-3.4) 3.6 (2.6-4.1) 3.4 (2.4-3.7) 3.7 (3-4.3) 3.4 (2.6-3.9) 0.069 occupational risks and hazards 2.7 (2.2-3.5) 3 (2.1-3.7) 2.8 (1.8-3.5) 2.6 (2-4) 2.8 (2-3.6) 0.988 conflicts and poor communication 2.9 (2-3.5) 3 (2.1-4) 3 (2-3.8) 3.3 (2.3-4.3) 3 (2-3.8) 0.368 shift work 2.8 (2-3.8) 3 (2.3-4.2) 3.3 (1.7-4) 3.7 (2.3-4.3) 3 (2-4) 0.451 professional requirements 3 (2.4-3.3) 3 (2.3-3.5) 3.2 (2.2-3.8) 3.4 (2.8-3.8) 3 (2.4-3.6) 0.368 total rating 3 (2.5-3.4) 3.2 (2.8-3.8) 3.3 (2.5-3.8) 3.5 (2.8-3.8) 3.2 (2.6-3.7) 0.264 * mann-whitney test according to the participants with secondary education qualifications, the greatest source of stress lies in work organisation and financial issues. there were no statistically significant differences based on the level of education (table 6). seemedj 2018, vol 2, no. 2 stress and stressors for surgical nurses 54 southeastern european medical journal, 2018; 2(2) table 6. average rating on stress scale by level of education median (25% 75%) by level of education p* secondary college university total work organisation and financial issues 3.5 (3-3.9) 4 (3.2-4.5) 3.2 (3.1-4.4) 3.5 (3.1-4) 0.051 public criticism and litigation 3.4 (2.6-3.8) 3.4 (2.7-4.1) 2.7 (1.9-4.1) 3.4 (2.6-3.9) 0.607 occupational risks and hazards 2.8 (2.2-3.6) 2.6 (1.4-3.6) 2.6 (1.64.1) 2.8 (2-3.6) 0.800 conflicts and poor communication 3 (2.3-3.8) 3.3 (2-4) 2.5 (1.9-4.4) 3 (2-3.8) 0.974 shift work 3 (2-4) 2.3 (1.7-4.3) 4 (3.3-4.5) 3 (2-4) 0.240 professional requirements 3 (2.4-3.6) 3 (2.2-3.6) 3.2 (2.7-4.4) 3 (2.4-3.6) 0.626 total rating 3.2 (2.7-3.6) 3.1 (2.5-4.1) 3 (2.5-4.2) 3.2 (2.6-3.7) 0.963 * mann-whitney test participants with the shortest length of service (up to 10 years), rated work organisation and financial issues as the most significant sources of stress were, whereas occupational risks and hazards were rated as the least significant sources of stress by the participants with 30 or more years of service (table 7). table 7. average rating on stress scale by length of service median (25% 75%) by the length of service p* up to 10 years 11 20 21 – 30 30 years and over total work organisation and financial issues 3.5 (3-3.6) 3.6 (3.1-4) 3.6 (3.2-4) 3.9 (3.5 4.1) 3.5 (3.1-4) 0.144 public criticism and litigation 2.9 (2.6-3.6) 3.3 (2.7-4) 3.5 (2.7-3.8) 3.7 (2.9 4.1) 3.4 (2.6-3.9) 0.229 occupational risks and hazards 2.8 (2.2-3.4) 2.8 (1.83.6) 2.7 (2-3.9) 2.8 (2.1 3.7) 2.8 (2-3.6) 0.997 conflicts and poor communication 2.8 (2-3.5) 3 (2.5-4) 3 (2-4) 3.3 (2.6 4.1) 3 (2-3.8) 0.258 shift work 2.7 (2-4) 3 (2-4) 3.7 (1.6-4.3) 3.3 (2.5 4.2) 3 (2-4) 0.634 professional requirements 3 (2.4-3.2) 3 (2.4-3.6) 3.3 (2.4-3.9) 3 (2.6 3.7) 3 (2.4-3.6) 0.275 total rating 3 (2.5-3.5) 3.2 (2.7-3.7) 3.4 (2.5-3.9) 3.5 (3 3.8) 3.2 (2.6-3.7) 0.316 * mann-whitney test seemedj 2018, vol 2, no. 2 stress and stressors for surgical nurses 55 southeastern european medical journal, 2018; 2(2) discussion the results of our research indicate that the participants working in operating rooms and intensive care units are exposed to a wide range of work stressors. golubić et al. (2009) identified the six following major groups of occupational stressors among croatian nurses: organisation of work and financial issues, public criticism, occupational hazards, interpersonal conflicts at workplace, shift work and professional and intellectual demands (16). due to a lack of staff, nurses who take care of patients come under more pressure. as a result, such pressure causes concern and stress for nurses, whereas the lack of staff causes an excess of administrative tasks and overtime work, usually not paid by the state (17). despite the lack of staff, the job has to be done, which leads to fatigue and exhaustion, thus further increasing the level of stress and keeping nurses in an everexpanding vicious cycle. it has been found that temporary contract nurses suffer from higher stress levels than permanent contract nurses (9). all around the world, nurses have recognised a small number of workers as one of their greatest problems (18). in the countries in transition, there has been an increase in the number of nurses leaving the nursing profession (19). work organisation and financial issues are significantly more stressful for nurses in comparison to any of the other factors mentioned in the research (20). the most common causes of stress for chinese nurses are the imbalance between investment and gain, poor image of nursing in society and organisational issues (21). when it comes to public criticism and litigation, the main stressors are constant responsibilities during 24-hour shifts, inappropriate public criticism and the threat of litigation. women are exposed to significantly higher levels of stress, above all because of public criticism and litigation. it has been suggested that such higher stress levels result from multiple and complex roles that women have to perform – the role of an employee, wife, mother and housekeeper (22). also, in a research conducted among croatian nurses, knežević (2009) pointed out that the stressors related to public criticism are the most common stressors for nurses working in non-surgical professions, followed by those working in surgical professions (23). regarding occupational risks and hazards, the most significant stressors are coping with incurable patients, fear of infection and fear of injury from sharp objects. these are the main sources of stress for nurses and the situation has not changed even after thirty years (24). in a research conducted among croatian nurses, golubić et al. (2009) concluded that nurses with secondary education qualifications perceived occupational hazards as statistically significantly more stressful than nurses a with college degree, as well as that offering educational and career prospects can contribute to decreasing nurses' occupational stress levels (16). conflict between nurses and health care staff includes the power of superiors, the ability to work in a team, interpersonal relations and skills and expectations at different levels (25). a study on nurses conducted in iran presented the factors that have an impact on conflicts (the nature of work, working conditions, work environment, hospital structure, management style and individual characteristics of employees) and that can improve or worsen the situation. the majority of nurses pointed out that they suffer from psychological stress due to misunderstandings between nurses and other staff (26). it is believed that good communication skills are required to create an effective relationship, which is why a lack of such skills can have a negative effect on nurses and their colleagues at work (27). certain personality traits can also affect occupational stress. “screening” before employment, used for identification and presentation of one’s “personality” and stress perception, can be considered a part of a particular employment strategy aimed at resolving the issues related to stress, illness and absence from work (28). the most common factors causing burnout are as follows: work overload, lack of control, insufficient rewards, poor labour relations, injustice and conflict between values (29). a research in belgium from 2015 showed that besides the softer work characteristics – such as seemedj 2018, vol 2, no. 2 stress and stressors for surgical nurses 56 southeastern european medical journal, 2018; 2(2) decision latitude, social capital and team cohesion – more insight and knowledge of the hard work characteristic workload is essential (30). education, training and career opportunities can reduce occupational stress experienced by nurses and at the same time contribute to maintaining their working capacity (31). overtime, shift and night work, responsibility in decision-making, contact with patients and their families and emotional exhaustion (burnout) of health care workers contribute to increased morbidity from mental disorders and psychosomatic diseases (32). in surgery, cognitive symptoms of stress include problems in concentration and memory, resulting in a risk for a patient’s safety (33). a study conducted in china has also indicated that a lack of nurses, excessive paperwork, increased number of operations, imposed time limits and night shifts are factors associated with stress that have a negative impact on patient care (34). serious problems and difficulties in patient care may cause crises in nurses’ work and lead to various stressors, such as criticism at work, crisis between superiors and subordinates and job dissatisfaction. all of the above leads to symptoms related to cardiovascular, digestive and musculoskeletal changes as sources of stress (10). overall, the greatest sources of stress for the participants in the study are work organisation and financial issues, with shift work as the most significant stressor for nurses with college education qualifications. occupational risks and hazards are the sources of stress for respondents aged 31-40. although some variations were observed, they were not statistically significant. a study of causes of stress among nurses in iran showed that workrelated factors were more relevant in causing occupational stress than demographic and other factors (35). however, a study conducted in serbia yielded opposite results (36). besides that, much research conducted among nurses has also shown show that there is a connection between particular diseases and work-related stress, such as emotional exhaustion (37), physical exhaustion (32) and lower back pain (38). conclusion in the summary of the results of this research, we conclude that total rating on the stress scale was 3.2 (interquartile range from 2.6 to 3.7). the most common stressors among surgical nurses are insufficient number of employees, work overload, administrative work, constant responsibilities during 24-hour shifts, inappropriate public criticism, fear of infection, conflicts with superiors, night and overtime work and pressure of imposed time limits. results showed no statistically significant differences in the level of stress with regard to the participants’ age, total length of service and level of education. regarding female respondents, a considerable amount of stress arises from public criticism and litigation. better work organisation, supportive work environment, continuous education and stress management programs are essential for critical care and operating room nurses. based on the above-mentioned facts, it is clear that further research on this matter must be conducted. acknowledgement. none disclosure funding. no specific funding was received for this study. competing interests. none to declare references 1. ilo standards-related activities in the area of occupational safety and health. in: international labour conference, 91st session, report vi. p.31-8. geneva, switzerland. ilo, 2003. 2. mcgrath jj, prochazka j, pelouch v, oštadal b. physiological responses of rats to intermittent high-altitude stress: effects of age. j appl physiol 1973; 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8(6): 386-93. www.ncbi.nlm.nih.gov/pubmed/12539798 seemedj 2020, vol 4, no. 1 laboratory animal welfare approach in science 102 southeastern european medical journal, 2020; 4(1) short review article laboratory animal welfare approach in science 1 iris broman* 1 1 head of animal facility, faculty of medicine, josip juraj strossmayer university of osijek, croatia corresponding author: iris broman, iris.broman@mefos.hr received: sep 17, 2020; revised version accepted: jan 7, 2020; published: apr 27, 2020 keywords: laboratory animal, animal welfare, 3rs abstract the term laboratory animal welfare has been generating a huge amount of controversial questions in science since the very beginning. humans use animals that are suited to their needs, be they of a psychosocial, therapeutic, official, scientific or nutritional nature. due to their similarities to human and short life span, the percentage of rodents used for science will correspond to the percentage of similarity between our respective genomes. the era of genetic manipulation led to accelerated and unnatural manipulation of life forms, which was quite shocking for the generations of the last millennium. yet, it is completely normal today that genetic engineering is applied to living creatures or virtually. biomedicine is looking for insights into mechanisms of health preservation, clarity when it comes to development of diseases, prevention of and treatment methods for diseases, researching the environment, and improving the quality of life for mankind. science observes the phenomena of life in action, and animals change quickly, which makes them appropriate for our objectives. the sheer number of chosen animals with known parameters and bodily responses that can be used in biostatistical analysis, which are expected to help us obtain our results, calls for an approach to animal welfare in science. alternative methods are being developed through teamwork of researchers and veterinarians in order to find methods that will overall decrease animal pain and suffering to the lowest degree possible, while increasing the possibility of valuable research contributions. legislative background is available as a starting point and gives researchers basic guidelines for animal welfare categorization. (broman* i. laboratory animal welfare approach in science. seemedj 2020; 4(1); 102-105) seemedj 2020, vol 4, no. 1 stigmatization of patients with viral infections and mass psychogenic illness 103 southeastern european medical journal, 2020; 4(1) laboratory animal welfare approach in science the term laboratory animal welfare has been generating a huge amount of controversial questions in science since the very beginning. the idea that a being deliberately changes another being, who is at present healthy and content, into a being that is not sounds controversial as such. on the other hand, one must ask oneself if laboratory animal science is in fact the worst possible mode of animal exploitation for human purposes. the attitude depends greatly on the situation in which the thinker finds themselves and on their knowledge of the animal’s actual needs, ensuring that human demands are fulfilled, factors that justify man’s involvement and money that one spends on animal maintenance, care and breeding. animals do not need humans. if humans had never appeared on the evolutionary tree, a healthy assumption is that none of the animals would ever think: “if only there were people around!” we are the ones who need them; we have absolute control over their exploitation and we place them in isolation from others of their kind in conditions that are suited to our needs, be they of a psychosocial, therapeutic, official, scientific or nutritional nature. if we take a shortcut from synapsids (323 million years ago) to the cretaceous-paleogene extinction event (around 66 million years ago), which wiped out most of ectothermic/poikilothermic organisms on earth, there was a little, primitive rodent that somehow managed to survive (1). later on, it spread all over the earth, evolved and, providing that we subscribe to the theory, became human. the most likely relative (protungulatum donnae) of these ancient humans (1) had a placenta and all other parts that contemporary rodents have, as well as the rest of the animals that we share common ancestry with, which are genetically similar to us. their bodily responses, reproduction methods, weaning, instincts, curiosity, and nutritional needs are quite comparable to ours. a species that kept its pleistocene form until today, and may also be a direct intact living ancestor to our own species is the endemic species, the balkan snow vole (2). the balkan snow vole today inhabits high-altitude areas of the dinarides, in croatia among other countries, and is categorized as “vulnerable” in iucn's red list of threatened species (3). today, as will most likely also be the case for many years to come, the percentage of small rodents used for science will correspond to the percentage of similarity between our respective genomes. by using selection, we have gained absolute control over the majority of species, in fact over all species that were of any interest to us; it further changes according to fashion and refinement of methods, as well as upgrading of breeding methods (4, 5). the era of genetic manipulation led to accelerated and unnatural manipulation of life forms, which was quite shocking for the generation of the last millennium, yet it is completely normal today that genetic engineering is applied to living creatures or virtually. if we follow the crossroads towards the science branch, there is a rise of multiple interested parties in search of their own object of research. biomedicine is looking for insights into mechanisms of health preservation, clarity when it comes to development of diseases, prevention of and treatment methods for diseases, researching the environment, avoiding and preventing the spreading and development of factors that can lead to disease, improving the quality of life for mankind. a pathological state is any state that differs from the homeostatic state, which would fit the age, gender, psychophysical factors and uninhibited possibility of movement, with the ability to fulfill the basic needs of life. science requires living animals in order to understand the mechanisms that are preventing those differences. in order to discover as many reasons behind such differences as possible, one must understand the functions of various mechanisms, as well as the integration of said mechanisms on different levels. molecular mechanisms are constantly in an active state of integration, power and separation in certain cycles, influences and feedbacks. scientists stick to the parameters seemedj 2020, vol 4, no. 1 stigmatization of patients with viral infections and mass psychogenic illness 104 southeastern european medical journal, 2020; 4(1) that they can measure and explain by the known laws of physics, biology and other branches of science that observe the phenomena of life in action. by observing life in action, researchers develop methods as tools to prove the accuracy of their hypotheses, which proof also has to be reproducible and applicable to certain mechanisms or groups of mechanisms that are present in their current field of work. by developing methods, scientists produce results, or more accurately, enhance the clarity of further control and manipulation of changes, which can have a larger or smaller impact on the welfare and sustainable development of humanity. laboratory animals are placed in the category of materials used in biomedical research. studying their body mechanisms and inducing controlled changes in them by using different methods leads to various scientific contributions. the choice of certain species, which are chosen by selection methods or by direct influence on their genome, is described in research protocol. competent bodies and institutions evaluate material, financial institutions decide whether and to which degree obtained results are worth their while. if they are, the project is approved. justification for extracting the animal from nature (it does not have to be a green or snowy picturesque landscape, it could be the city sewer system, if the purpose is to collect ecological samples) calls for animal welfare in science (6). a controlled lab experiment is done with a familiar animal with a health and breed certificate and produces definite results. methods like gene modification, drastic, fast, aim applicable or slower selective methods, number of chosen animals with known parameters and bodily responses that can be used in biostatistical analysis, are expected to give us purpose in our results. animal welfare can be approached in numerous ways; it has applicable and non-applicable measurements of value, it promotes various debates, inspires blogs and it’s even used as a cover for terrorist attacks (7). the role of veterinary medicine does in biomedical science is to ensure that the animals are healthy and the environment is as harmless as could be at the given moment, and it tries to engage in activities with the aim of decreasing the damage in a given situation for all parties involved. this means developing methods through teamwork with competent researchers in order to find methods that will decrease animal pain and suffering to the lowest degree possible, while increasing the possibility of valuable research contributions. the scientific and professional approach attempts to preserve the balance and the degree to which genetic modifications are used in order to achieve scientific results. the same balance is of use to 3rs (9) (reduction, replacement, refinement in animal research), so genetics in biomedical research gets huge points in animal welfare, as well. geneticsrelated research serves the purpose of developing healthcare, including prevention, diagnostics and treatment, as does the occasional funding for that scope of projects. from now on, every scientist who declares laboratory animals as a necessary material for their work and cannot achieve their result by using known alternative methods, needs to start with research and personal progress now and consider animal welfare from the start. this offers the possibility of involvement in the right way and through proper channels, because, during future research, it will lead to the work having far better quality, direction and recognition in the end. there are unavoidable guidelines on animal welfare sites, which are being refined on a daily basis and which help researchers express the integrative plan of future research. refined sites list legislative background to start from and give the researcher the basic starting guideline categorization. .acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. seemedj 2020, vol 4, no. 1 stigmatization of patients with viral infections and mass psychogenic illness 105 southeastern european medical journal, 2020; 4(1) references 1. archibald jd, zhang y, harper t, cifelli richard l. protungulatum, confirmed cretaceous occurrence of an otherwise paleocene eutherian (placental?) mammal. j of mammal evol. 2011;18: 153–161. doi:10.1007/s10914-011-9162-1. 2. kryštufek b., bužan e. rarity and decline in palaeoendemic martino’s vole dinaromys bogdanovi, mammal review. 2008; 38(4): 267284. 3. the iucn’s red list of treatened species. https://www.iucnredlist.org/ (retrieved may 13, 2019) 4. zhang f, li d, wu q, sun j, guan w, hou y, zhu y, wang j. prepartum body conditions affect insulin signaling pathways in postpartum adipose tissues in transition dairy cows. j animal sci biotechnol 2019; 10: 38. https://doi.org/10.1186/s40104-019-0347-4 5. hayes bj, bowman pj, chamberlain aj, savin k, van tassell cp, sonstegard ts, goddard me. a validated genome wide association study to breed cattle adapted to an environment altered by climate change plos one 2009; 4(8): e6676. https://doi.org/10.1371/journal.pone.0006676 6. guide for the care and use of laboratory animals 8th ed. committee for the update of the guide for the care and use of laboratory animals, institute for laboratory animal research, division on earth and life studies, national research council of the national academies. publisher: the national academies press, washington, d.c https://grants.nih.gov/grants/olaw/guide-forthe-care-and-use-of-laboratory-animals.pdf (retrieved january 31, 2020) 7. u.s. code, title 18. crimes and criminal procedure part i. crimes chapter 3. animals, birds, fish, and plants section 43. force, violence, and threats involving animal enterprises. https://www.law.cornell.edu/uscode/text/18/ 43 (retrieved january 31, 2020) seemedj 2019, vol 3, no. 1 quality of life in patients with malignant disease 1 southeastern european medical journal, 2019; 3(1) original article quality of life in patients with malignant disease 1 suzana stojković1, nada prlić,2 1 department of cardiac surgery, surgical clinic, university hospital center, osijek, croatia 2 josip juraj strossmayer university of osijek, faculty of medicine osijek, department of nursing, medical ethics and palliative medicine, osijek, croatia corresponding author: suzana stojković, stojkovic.suzana@gmail.com received: august 29, 2017; revised version accepted: may 7, 2019; published: may 31, 2019 keywords: patients, quality of life, malignant disease abstract aim: to assess the quality of life in patients with a malignant disease. research subjects and methods: research included 105 patients with a malignant disease who were receiving stationary and daily treatment at the radiotherapy and oncology department of the university hospital center, osijek, croatia. a questionnaire containing various demographic data and including a scale for measuring the quality of life in patients with a malignant disease – the functional assessment of cancer therapy-general (fact-g) – was used as a research instrument. results: average score on the scale was 89. level of satisfaction with social/family relationships was significantly lower in older respondents (p = 0.027), single persons (p = 0.018) and participants with total income under hrk 3,000 (p = 0.031). regarding family and social relationships, the patients receiving hospital day care expressed a significantly higher level of satisfaction (p = 0.001), as well as the subjects with college/university qualifications (p = 0.007). patients with malignant disease of the head and neck expressed significantly lower levels of satisfaction on all subscales and with regard to overall health (p = 0.005). conclusion: quality of life in patients with a malignant disease is satisfactory. (stojković s, prlić n. quality of life in patients with malignant disease. seemedj 2019; 3(1); 1-10) seemedj 2019, vol 3, no. 1 quality of life in patients with malignant disease 2 southeastern european medical journal, 2019; 3(1) introduction quality of life is an extremely complex concept involving various scientific disciplines. given the complexity and number of perspectives that the concept reflects on, it is almost impossible to provide an unambiguous definition of the quality of life (1). its definitions vary, just as the manner in which it is assessed. raphael et al. state that the assessment method can also influence the definition of the quality of life. whereas a medical approach emphasizes the impact of illnesses and difficulties on the quality of life, an approach focusing on health draws attention to well-being and the abilities required in everyday life (2). researchers agree that the concept of quality of life is multidimensional and subjective (3). krizmanić and kolesarić state that the quality of life is a subjective experience of one's own life determined by objective circumstances in which a person lives, personality traits affecting the experience of reality and the person’s specific life experience (4). the concept of quality of life in each person relates to the experience of satisfaction with the way of life, its course and conditions, perspective, possibilities and limitations (5). felce and perry define the quality of life as an overall, general well-being including objective factors and a subjective evaluation of physical, material, social and emotional well-being. it includes personal development and purposeful activity valued through a personal set of values. they also accentuate the linkage of objective and subjective indicators. it has been found that there is a weak connection between a person’s subjective feeling of life satisfaction and a selfassessment of the quality of life and objective living conditions (6). a significant correlation of subjective and objective indicators has been found in situations of poverty and misery, when basic human needs are not met. regarding life in developed countries, some authors stated that “people in the 1990s are, on average, four and a half times richer than their ancestors from the beginning of the 20th century but are not four and a half times happier” (7). cella and tulsky presented the four basic dimensions of the quality of life: physical, business, emotional and social well-being (8). patients diagnosed with cancer often experience a reduction in the quality of life to a variable degree, but they can adapt to life with cancer (9). today, the therapeutic effects in the treatment of patients should no longer be compared and evaluated only on the basis of improvements in laboratory findings and survival of patients, but also on the basis of improvements in their quality of life (10). despite a great progress in the treatment of malignant diseases, the course of illness, treatment and psychological challenges have a negative impact on a patient’s quality of life (11). being diagnosed with a malignant disease is in itself stressful. apart from excessive physical stress caused by illness and its treatment, many patients also experience psychological stress and worry about their diagnosis and prognosis, difficult treatments and decisions, as well as the change in common life functions and roles. a recent study conducted in the united states has shown that long-term cancer survival increases serious psychological difficulties among those who have been disease-free for five-years compared to the general population (12). malignant disease can lead to major life-style changes; it can cause serious disorders in a person's normal functioning, from illness to severe financial crises (13). it has been found that 33% of individuals diagnosed with cancer experience psychological pain, whereas up to 70% of cancer patients experience some degree of anxiety and depression (14). regardless of treatment, these individuals experience changes in their physical and emotional integrity, restlessness, pain, changes in appearance, dependence on others and loss of self-confidence, which consequently reduces their quality of life in a short time (15). people living with malignant diseases have a greater risk of developing various psychological problems. studies have shown that such patients suffer not only from physical symptoms of a disease, but also from psychological and social stress associated with the diagnosis. seemedj 2019, vol 3, no. 1 quality of life in patients with malignant disease 3 southeastern european medical journal, 2019; 3(1) research has also shown that, apart from the fear of dying, patients feel endangered by interventions, such as chemotherapy or radio therapy, and they worry about losing their physical integrity, independence and social roles (16). prolonging a patient’s life has always been one of the dominating objectives of traditional medicine. as the success in this direction has increased, it has become clear that such a goal is inadequate. medicine must strive not only to add years of life, but also help ensure the quality of life acceptable and worthy of human being for the years added (15). incidence of malignant diseases (cancer) varies across the world and has been changing over time. cancer is a major public concern in the entire world. in developed countries, it is often the second leading cause of death after cardiovascular disease. according to the latest international data, 12.7 million new cases are discovered annually, 7.6 million people die of cancer, and 28.0 million people live with cancer within 5 years of diagnosis. cancer is also the second most significant cause of death in croatia, from which every fourth resident dies. cancer incidence rates by the 15 most common primary sites in osijek-baranja county in 2013 was 472.5/100,000 for men and 383.8/100,000 for women. also, cancer incidence rates in croatia have been increasing (17). the main objective of this study was to examine the quality of life in patients with a malignant disease. specific goals were focused on the following issue: examining whether there is a difference in the quality of life in patients with malignant diseases regarding the type of malignant disease, age, gender, education level, marital status, financial situation and place of treatment (day hospital, stationary treatment). research subjects and methods research subjects were patients suffering from malignant diseases who were receiving stationary treatment and those treated in the day hospital at the department of radiotherapy oncology, osijek university hospital center. there were 105 respondents participating in the study, of whom 71 were treated in a day hospital and 34 were ward patients. research was conducted from 18 august to 30 october 2015. a survey questionnaire containing various demographic data (age, gender, level of education, illness, marital status, and financial situation) was used as a research instrument. functional assessment of cancer therapygeneral (fact-g) scale was used for the selfassessment of the quality of life in malignant patients. fact-g questionnaire contained 27 particles. it evaluated the four following areas: physical well-being, social/family well-being, emotional well-being and functional well-being. physical well-being was covered by gp particles (gp 1, 2, 3, 4, 5, 6, 7), social/family well-being was covered by gs particles (gs 1, 2, 3, 4, 5, 6, 7) and emotional benefit was included in ge particles (ge 1, 2, 3, 4, 5, 6). functional benefits were covered by gf particles (gf 1, 2, 3, 4, 5, 6, 7). the answer for each particle was evaluated on the likert scale of 0 – 4 (0 – not at all, 1 – a little, 2 – somewhat, 3 – a lot, 4 – very much). overall result on the whole scale was 0 – 108. total result for the area of physical and social/family well-being could range from 0 to 28, whereas the result for the area of emotional and functional well-being could range from 0 to 24. the assessment of the quality of life related to seven days prior to the testing day. in all areas of the questionnaire, higher results represented better quality of life (9). in a literature review, victorson et al. (2008) reported cronbach’s alpha reliability coefficients from about 78 published studies. average fact-g score reliability was .88 and subscales ranged between .71–.83 (ref). internal consistency for the sample of participants in this research was very good (cronbach's alpha 0.72 – 0.85) (18). statistical analysis category data was represented in absolute and relative frequencies. numerical data was described by the median and the interquartile range limits. seemedj 2019, vol 3, no. 1 quality of life in patients with malignant disease 4 southeastern european medical journal, 2019; 3(1) normality of the distribution of numeric variables was tested by kolmogorov-smirnov test. differences between normally distributed numeric variables between the two independent groups were tested by the mannwhitney u-test, and, in the case of 3 and more groups of independent groups, by the kruskalwallis test. due to a deviation from the normal distribution, the correlation was estimated by the spearman coefficient of correlation ρ (rho). all p values were two-sided. level of significance was set to α = 0.05. statistical analysis of the data was done by the statistical program spps for windows (version 16.0, spss inc., chicago, il, usa). ethical principles prior to the research, a written consent of the commission for ethical and vocational issues of nurses at the osijek university hospital center was obtained on 29 june 2015, as well as a written consent of the head nurse of the department of radiotherapy oncology of the osijek university hospital center. all respondents were informed about the purpose of the research. they received a written notice for respondents, a document on consent and a statement of consent of the informed interviewee to participate in the research. they all willingly agreed to participate in the research, which they confirmed by their signature. research was conducted in accordance with ethical principles and human rights in research. results study was conducted on 105 respondents with a malignant disease, of whom 71 (67.6%) were treated in a day hospital, and 34 (32.4%) were patients receiving stationary treatment. average age of respondents was 62 (interquartile range of 55-71), ranging from 33 to 88 years of age. there were more male than female respondents – 58 (55.2%). regarding the level of education, 60 (57.1%) of respondents had vocational qualifications. there were 71 (67.0%) married respondents and 93 respondents (88.6%) reported having children. eight subjects (8.0%) had under hrk 3,000 of monthly household income, 55 (55.0%) of them had monthly household income in the amount of hrk 3,000 to 5,000 and only two respondents had income ranging from hrk 12,000 to 20,000. the most common site of malignant disease was the thoracic region and digestive tract. with regard to whether the subjects were being treated in a day hospital or receiving stationary treatment, the mean values of single particles and of the overall scale were equal, with the exception of social/family relationships: the patients treated in the day hospital responded significantly better – the median was 24.0 (interquartile range of 22.2 to 26.8) (p = 0.001). the mean value on the overall fact-g scale was 89.0 (interquartile range of 73.3 to 95.5). the lowest rating on the overall scale was given by those with a lower education level – the median of 80.5 (interquartile range of 64.8 – 94.2). overall satisfaction was significantly higher in subjects with a college or higher education level – the median of 98.5 (interquartile range 87.1 to 101.8) (p = 0.007). regarding the particles, these subjects were more satisfied with their physical health (p = 0.009), social/family relationships (p = 0.032), and functional state (p = 0.016), while the emotional state was equal regardless of the level of education (table 1). seemedj 2019, vol 3, no. 1 quality of life in patients with malignant disease 5 southeastern european medical journal, 2019; 3(1) table 1. quality of life regarding education level subscales median (interquartile range) regarding education level p* unqualified vocational college or graduate total physical health 21.5 (15.5 25.3) 23 (18.2– 26.0) 26.5 (22.1– 28.0) 23 (18.0– 26.0) 0.009 social/family relationships 22.1 (17.9 26.2) 23 (21.0– 25.0) 25.1 (24.0 26.9) 24 (21.0 25.9) 0.032 emotional state 19 (12.0– 22.0) 21 (18.0– 23.0) 22 (18.8 23.3) 21 (17.0– 23.0) 0.103 functional state 20 (16.0 24.3) 21 (16.0– 25.0) 25 (21.0– 27.0) 21 (17.0– 25.0) 0.016 overall fact-g scale 80.5 (64.8 94.2) 89 (74.2– 95.0) 98.5 (87.1 101.8) 89 (73.3 95.5) 0.007 * kruskal-wallis test patients with malignant disease of the head and neck reported the lowest satisfaction with their physical health (p = 0.004), social/family relationships (p = 0.003), emotional state (p = 0.044), functional state (p = 0.021) and overall health (p = 0.005) (table 2). table 2. quality of life regarding site of malignant disease subscales median (interquartile range) regarding site of malignant disease p* head and neck thoracic region digestive tract uro-genital tract total physical health 17.5 (16.0– 21.0) 24.5 (20.0– 26.0) 21 (15.5 25.0) 26 (22.8 27.3) 23 (18.0– 26.0) 0.004 social/ family relationships 18 (16.0 22.2) 24 (21.0 26.8) 23 (21.0 24.7) 24.2 (22.8 25.4) 24 (21.0 25.9) 0.003 emotional state 14 (12.0– 22.0) 21 (19.0– 23.0) 18 (15.0 22.5) 21 (20.0 22.5) 21 (17.0– 23.0) 0.044 functional state 16 (13.0– 20.0) 22 (19.0 25.2) 20 (17.0 24.5) 21 (19.5 – 24.0) 21 (17.0– 25.0) 0.021 overall factg scale 66.5 (53.0 81.2) 90.4 (83.8 97.3) 84 (72.0 94.9) 92.8 (84.5 96.3) 89 (73.3 95.5) 0.005 * kruskal-wallis test seemedj 2019, vol 3, no. 1 quality of life in patients with malignant disease 6 southeastern european medical journal, 2019; 3(1) respondents up to 50 years of age were significantly more satisfied with social/family relationships – the median of 26 (interquartile range of 23.2 to 28.0). there were no significant differences in other subscales (p = 0.027). areas of the scale have a significantly positive correlation (p < 0.001). discussion according to the research literature, the most common cancer site was the thoracic region, which is in accordance with our research results. most respondents had secondary education qualifications, were married and had average income in relation to the standard of our country. the number of deaths due to prostate cancer has been increasing (19). this is also apparent from our research, which found that malignant diseases of the urogenital tract are the third most frequent diseases. regarding physical health, the majority of respondents, 59 (57.8%) of them, stated that they had energy. on the other hand, it is interesting to note that a large number of respondents, 24 (23.5%) of them, were quite or very much lacking in energy. such a finding was expected considering the nature of the disease, especially of malignant diseases in lung and thoracic regions, as described in the available literature (20). it is also interesting that patients can hardly meet the expectations of their families. this may point to an inadequate psychological assistance provided to cancer patients and their families. psychological support for patients and their families is extremely important when they go through the shock and stress of being confronted with a diagnosis and being in the state of fear, loneliness and demoralization between therapies, as well as in critical situations when their condition worsens and prognoses are not optimistic. support can be offered by the persons whom the patients trust and with whom they are close. family and friends are play a vital role in providing patients with boost and help (5, 21). this is also apparent from our research, which shows that for the largest number of respondents, a great amount of support comes from their family or a person close to them, most commonly a partner. the majority of the respondents agreed that their friends were giving them a lot of psychological help and support. sexuality, which unfortunately receives very little attention, is a major issue for severely ill patients. health care staff is so busy that there is no time to ask the patient about their sex life. such discussions are often confusing for patients, or they are lacking sufficient knowledge to provide an answer to the questions asked. most people in our community consider talking about sex life indecent. it is still a taboo topic, even in conversation with healthcare staff (22). the majority of subjects did not want to answer the question of whether they were satisfied with their sex life, while 29 of them (40.3%) responded that they were barely or not satisfied with their sex life. it is apparent from our research that a number of respondents were lacking in sexual rehabilitation, which can no longer be a matter of choice rather than a need. most of our respondents were sleeping well and enjoying things they do for entertainment. compared to other researchers who have dealt with the same topic, our results did not deviate from the values of their results. the results support the fact that physical activity and the inclusion of oncological patients and their families in the community contribute to a more successful outcome of rehabilitation, the objective of which is an increase in the quality of life (5). the overall value of the quality of life in patients with a malignant disease in our study was 89 (fact-g), which is similar to the results of other studies (8, 5). the majority of research has shown that patients with a malignant disease have a good quality of life. good social and family relationships certainly have a significant impact on the quality of life of our respondents. a higher level of satisfaction with the quality of life has been found in highly educated subjects. higher education leads to a better knowledge about the illness and the manner of coping with it, as well as to increased availability of seemedj 2019, vol 3, no. 1 quality of life in patients with malignant disease 7 southeastern european medical journal, 2019; 3(1) information about the treatment and psychological self-help. in the martinis’ research, the results have shown that men aged between 50 and 59, younger people and people with higher education qualifications are more satisfied in all areas of the quality of life (23). results of a research conducted on a large group of australian women, three and twelve months after early breast cancer surgery, have shown that the impact of illness and treatment on the quality of life varies according to age, education and marital status. single women and those with a lower education level have rated their quality of life as bad in a number of particles (24, 25). croatian authors have also suggested that a better quality of life is associated with higher education. higher level of education is positively associated with a greater degree of satisfaction in certain areas of life, as well as with the achievement of patients’ goals, wishes and hopes (26). the lowest score was given by unmarried respondents, especially on the social/family relationships subscale. this is understandable in terms of the impact of family and friends on caring for such patients. the same results were also described in a study conducted in queensland, where married or cohabiting respondents had better results in the area of social-family relationships than unmarried respondents (8). a significantly lower level of satisfaction with the quality of life was found in respondents in less favourable financial situations. generally, it is expected that patients with a lower total income will be more dissatisfied with the quality of their life because treatment causes expenses due to which they are unable to provide everything they need for the best quality of life. patients with malignant disease of the head and neck are least satisfied with the quality of life. head and neck cancer is specific because the disease itself and the surgical removal of the tumour affect the most visible parts of the body and often have an adverse effect on daily activities, such as swallowing, breathing, speech and appearance (25, 27). respondents up to 50 years of age are significantly more satisfied with social/family relationships. with the increase of age, the level of satisfaction with social/family relationships decreases significantly, while the connection with other particles was weak. the overall value of the scale was also higher among the youngest group of respondents. this means that older patients are less satisfied with social/family relationships, i.e. they are emotionally and functionally dissatisfied. studies have shown that a family-oriented treatment of chronic diseases represents progress in treatment when compared to a traditional diseaseor patient-oriented treatment (5). patients want to be treated as persons with a character rather than persons with a malignant diagnosis. a person’s character implies a physical, emotional, and spiritual dimension. neglecting any of these human dimensions leaves the patient feeling empty and incomplete, which can result in a more difficult and slower recovery. that is certainly not the patient’s goal nor should it be the goal of the health care staff (28). the quality of life of patients with a malignant disease is satisfactory. there is a significant difference in the quality of life of respondents, in all areas of health, in relation to the site of a malignant disease. patients with malignant disease of the head and neck are considerably less satisfied with physical health, social/family relationships, emotional and functional state and with overall health. there is also a significant difference with regard to the age of respondents in the field of social/family relationships. the quality of life is lowest among the respondents aged 51 to 60. there is no significant difference in the area of health of respondents and the overall quality of life with regard to gender. there is a considerable difference in the quality of life regarding the level of education of respondents in the areas of physical health, social/family relationships and functional state. a significantly greater overall satisfaction with the quality of life was noticed among the respondents with college or higher education qualifications. seemedj 2019, vol 3, no. 1 quality of life in patients with malignant disease 8 southeastern european medical journal, 2019; 3(1) there is a significant difference on the subscale of social/family relationships with regard to the marital status of respondents. the highest rating on the overall quality of life scale was noticed in divorced respondents and the lowest in unmarried respondents, without statistically significant differences. a significant difference exists in the quality of life of respondents in the field of social/family relationships with regard to their financial situation. the lowest rating was given by the patients whose total income amounted to under hrk 3,000. there is a significant difference regarding the place of treatment in the subscale of social/family relationships. the patients treated in the day hospital are more satisfied with the quality of life. there is no recent data available for this geographic area on the subject of research. conclusion fact-g meets all requirements for use in oncological clinical trials, including ease of administration, brevity, reliability, validity and ability to respond to clinical change. selecting it for a clinical trial adds the capability to assess the relative weight of various aspects of ql from the patient's perspective. quality of life based on the health of people with a malignant disease must be the ultimate outcome of the rehabilitation process for each patient with a malignant disease. acknowledgement. i would like to thank all the departments at which this survey was conducted, as well as all participants in the research, for their cooperation. the paper was presented as a graduate thesis at the faculty of medicine of j. j. strossmayer university in osijek in 2016. disclosure funding. no specific funding was received for this study. competing interests. none to declare references 1. slavuj l. objektivni i subjektivni pokazatelji u istraživanju koncepta kvalitete života. geoadria. 2012; 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(seehsj), 2012; 2:52-61 seemedj 2019, vol 3, no. 2 hemoglobin a1c levels influences 76 southeastern european medical journal, 2019; 3(2) review article effect of iron deficiency anemia and other clinical conditions on hemoglobin a1c levels 1 domagoj vučić 1, karolina veselski 1, zvonimir bosnić ² 1 general hospital dr josip benčević, slavonski brod, croatia 2 health center slavonski brod, croatia corresponding author: domagoj vučić, domagojvucicmedri@gmail.com received: sep 1, 2019; revised version accepted: dec 5, 2019; published: dec 16, 2019 keywords: iron deficiency anemia, hemoglobin a1c, diabetes mellitus abstract iron deficiency anemia is the most common type of anemia in the world, the most common form of malnutrition deficit with a prevalence of 50% affecting the developed countries as well as developing countries with a strong influence on social and economic development. according to current guidelines of the american diabetes association (2019.) glycated hemoglobin (hba1c) is a reflection of the patient's glycemic status in the last three months and is used for monitoring of therapeutic effect as well as for diagnostic purposes. previous studies have proven that not only iron deficiency anemia but also a range of other clinical conditions can affect the level of hba1c independent of glycemic status. the exact mechanism of the effect of iron deficiency on glycated hemoglobin levels remains unknown and is still at the hypothesis level. studies have proven that treatment of iron deficiency anemia leads to better control of hba1c level, regardless of whether the patient is diabetic or not. a small number of studies have noted a correlation between iron deficiency and levels of glycated hemoglobin, thus further research on larger number of patients is certainly necessary in order to improve the therapeutic possibilities for patients with diabetes, more accurately diagnose and understand the pathophysiology of formation and influence on glycated hemoglobin levels. (vučić d, veselski k, bosnić z. effect of iron deficiency anemia and other clinical conditions on hemoglobin a1c levels. seemedj 2019; 3(2); 76-81) seemedj 2019, vol 3, no. 2 hemoglobin a1c levels influences 77 southeastern european medical journal, 2019; 3(2) introduction iron deficiency anemia in today's modern world is the most common form of malnutrition and the most common form of anemia. globally, iron deficiency is the cause of 50% of all anemias (1). hemoglobin a1c (hba1c) is a glycated hemoglobin, which is used to monitor average value of glycemic index of the patient in the last 3 months (2). hba1c is produced by glycation of the terminal amino acid valine at the β chain of hemoglobin and, unlike other markers for monitoring glycemic status, is less susceptible to daily fluctuations depending on diet, physical activity or disease (3). according to the latest guidelines of the american diabetes association (2019), the level of hba1c ≥ 6.5% is the criteria for the diagnosis of diabetes (3). various other factors can also affect glycated hemoglobin levels such as hemolytic anemia (4), hemoglobinopathies (5), acute blood loss (6), pregnancy (7) and uremia in chronic renal failure (8). what can be concluded is that the level of hemoglobin a1c is not only related to diabetes and glycemic index, but that different factors can affect the level of the same (table 1). table 1. diseases and clinical conditions affecting hemoglobin a1c level hba1c≥6.5% diabetes mellitus chronic renal failure uremia iron deficiency anemia hemoglobinopathies hemolytic anemia acute blood loss pregnancy note: summarized findings from studies to date on the most common diseases and clinical conditions that affect the value of hba1c levels. therefore, it is of the utmost importance to identify comorbidities that may affect the level of glycated hemoglobin before its level is taken into account for diagnostic and therapeutic purposes. new studies have provided data on alternative glycemic markers that are not as susceptible to these comorbidities and that can be used to monitor glycemic status in particular patient populations, but their application has not yet become widely used. from past to present previous studies which examined the correlation of sideropenic anemia and hba1c levels (depending or independent of the patient's glycemic status) have shown conflicting results. books and colleagues examined hba1c values in 35 patients with iron dificiency anemia without proven diabetes, and found significantly elevated levels of hba1c in patients with iron deficiency anemia and normalization of values after iron substitution therapy (9). similar results were obtained in the studies of sluiter et al. (10) and mitchell et al. (11) who attempted to explain the correlation of hba1c and sideropenic anemia by alternation of hemoglobin structure and hba1c levels in old and newly synthesized erythrocytes. according to the conclusion of sluiter et al., hemoglobin glycation is an irreversible process that is present in accordance with the erythrocyte lifetime. a prospective study from 2010, on a small sample of patients with proven diabetes mellitus and chronic renal failure (stage iiib-iv), examined the effect of intravenous iron supplementation and erythropoietin stimulating agents (esa) on hba1c values. both therapeutic choices led to a statistically significant decrease in hba1c (12). similar results were obtained in patients with diabetes without developed renal insufficiency (13-16). clever was the first study to test the hypothesis that intravenous administration of iron carboximaltosis leads to a reduction in hba1c levels in patients with type 2 diabetes and sideropenic anemia, thereby improving metabolic status and quality of life. organized as a randomized, single-blind study, it was conducted on subjects divided into two groups; study group (treated with iron carboximaltosis) and control group (treated with placebo). the seemedj 2019, vol 3, no. 2 hemoglobin a1c levels influences 78 southeastern european medical journal, 2019; 3(2) findings show that iron deficiency is associated with an increased risk of developing insulin resistance and obesity, and iron excess with the development of diabetes (the effect of pancreatic beta-cell stimulation on enhanced insulin secretion) (17). a 2016. cross-sectional study conducten on 150 patients (75 patients with iron deficiency anemia and 75 patients without anemia) examined hba1c values relative to the degree of sideropenic anemia. anemia severity was defined as mild anemia (hb 120-129 g/l for men and 110-119 g/l for women), moderate anemia (hb 90-119 g/l for men and 80-109 g/l for women) and severe anemia (hb <90 g/l for men and <80 g/l for women). an elevated hba1c was found in the patient with anemia in proportion to the degree of iron deficiency. upon completion of the study, an increased hba1c value was observed in a group of patients with iron deficiency anemia that correlated with the severity of the anemia (18). similar results have been reported in other studies (19-20). increased glucose during pregnancy is now a well-known risk factor for the development of perinatal complications of mother and child, so strict control of the glycemic index during pregnancy is of particular importance. pregnant women with known gestational diabetes have a significantly higher risk of developing type 2 diabetes at the end of pregnancy than pregnant women with normal glucose levels during pregnancy (21). studies have shown that hemoglobin a1c during pregnancy (the gold standard of glycemic control) is not a true reflection of the glycemic status due to frequent iron deficiency anemia (22-23). studies also noted that glycated albumin (ga) values were well maintained throughout pregnancy, suggesting that elevated glycated hemoglobin during gestation could be independent of glycemic index and that glycated serum albumin presents more accurate factor for glycemic control in pregnancy. furthermore, some studies found no correlation between hba1c values in patients with iron deficiency anemia and control group (24-25), other studies have reported lower values of hba1c in diabetic patients with sideropenic anemia compared to diabetics without anemia (26), while other studies found elevated hba1c values upon completion of substitution therapy in patients with iron deficiency anemia (unknown variables) (27). disclosure in conclusion, hba1c is widely used today as a diagnostic tool for diabetes and a means of controlling the patient's glycemic status. different clinical conditions and diseases may affect the erythropoiesis process with an impact on hba1c levels. numerous clinical studies, mostly based on a small number of patients, have confirmed that in the presence of iron deficiency anemia, hba1c levels are elevated even in non-diabetic patients with normal glycemic status (table 2). seemedj 2019, vol 3, no. 2 hemoglobin a1c levels influences 79 southeastern european medical journal, 2019; 3(2) table 2. results of studies examining the association between hemoglobin a1c and sideropenic anemia author and year of the study no. of subjec ts iron deficenc y anemia diabete s mellitus serum iron (μmol/l ) / ferritin (ng/ml) hemoglobin (g/l) hba1c % (examine d group) hba1c % (control group) shanti et al. 2013. 50 yes no no data / 3.68 106±1.4 7.6±0.5 5.5±0.8 alap l. christy et al. 2014. * 120 yes yes no data / 11.41 95.4 (male) / 93.7 (female) 6.87±1.4 5.65±0.69 bhardway et al. 2016. 50 yes no 2.7 / 7.51 61.8 6.60±0..1 5.48±0.56 rajagopal et al. 2016. 75 yes no 2.12 / 120.9 114.6 6.84±0.07 5.12±0.04 sinha et al. 2011. 50 yes no no data / 7.0 62 4.6±0.6 5.5±0.6 solomon et al. 2019. * 174 yes yes no data / no data 99.7 6.18±1.57 7.74±1.81 * control group; diabetic patients without iron deficiency anemia all numeric data expressed as mean values the expected elevated values of glycated hemoglobin in patients with diabetes are well correlated with diabetes control, therefore, in patients with controlled blood glucose levels, hba1c is expected to be less than 6.5% (3). the initial belief that the level of glycated hemoglobin is proportional exclusively to glycemic status, has been rejected. the mechanism by which iron deficiency anemia affects the level of hba1c remains unknown, largely based on hypotheses. from the foregoing, it is suggested that the iron status of the patient be taken into account before interpetation of the hba1c value, with or without proven diabetes. early diagnosis and treatment of iron deficiency anemia in patients with diabetes may improve glycemic control and delay the onset of complications (28). as there is not enough studies on a large sample from this still-unexplained pathophysiological mechanism, further research is needed in the future to elucidate the correlation of hemoglobin glycation and iron deficiency. precisely because the limitations of hemoglobin a1c are known today, there is great interest in the use of alternative glycemic markers. among the alternative markers that can be used as a complement to hemoglobin a1c is glycated albumin. as the level of glycated hemoglobin is dependent on red blood cell turnover, albumins have a shorter half-life, therefore indicating a glycemic index of the last 2-3 weeks. postprandial glucose values are also better in states with marked disease progression, such as in type 1 fulminant diabetes mellitus. restrictions on the use of ga are pathological conditions related to blood albumin levels such as nephrotic syndrome, liver disease, obesity, glucocorticoid administration, cushing's syndrome and hyperthyroidism. regardless of the known, routine administration of glycated hemoglobin has not yet taken root in clinical practice, which will require long-term prospective studies (29-31). seemedj 2019, vol 3, no. 2 hemoglobin a1c levels influences 80 southeastern european medical journal, 2019; 3(2) acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. john a. iron deficiency and other hypoproliferative anemias. in: longo d, fauci a, kasper d, hauser s, jameson j, loscalzo j, editors. principles of internal medicine by harrisons. 20th ed. united states of america: mcgraw-hill; 2018. pp. 684–90. 2. telen mj, kaufman re (2004) the mature erythrocyte. in: greer jp, forester j (eds). wintrobe's clinical hematology. (11thedn). lippincot: williams and wilkins 230. 3. american diabetes association diabetes care 2019 jan; 42(supplement 1): s13-s28. 4. horton bf, huisman th (1965) studies on the heterogeneity of hemoglobin. vii. minor hemoglobin components in haematological diseases. br j haematol 11: 296-304. 5. eberentz-lhomme c, ducrocq r, intrator s, elion j, nunez e, et al. (1984) haemoglobinopathies: a pitfall in the assessment of glycosylated haemoglobin by ion-exchange chromatography. diabetologia 27: 596-598. 6. starkman hs, wacks m, soeldner js, kim a (1983) effect of acute blood loss on glycosylated hemoglobin determinations in normal subjects. diabetes care 6: 291-294. 7. lind t, cheyne ga (1979) effect of normal pregnancy upon the glycosylated haemoglobins. br j obstetgynaecol 86: 210-213. 8. paisey r, banks r, holton r, young k, hopton m, et al. (1986) glycosylated haemoglobin in uraemia. diabet med 3: 445-448. 9. brooks ap, metcalfe j, day jl, edwards ms. iron deficiency and glycosylated haemoglobin a1.lancet. 1980 jul;316(8186) 10. sluiter wj, van essen lh, reitsma wd, doorenbos h (1980) glycosylated haemoglobin and iron deficiency. lancet 2: 531-532. 11. mitchell tr, anderson d, shepperd j (1980) iron deficiency, haemochromatosis, and glycosylated haemoglobin. lancet 2: 747. 12. ng jm, cooke m, bhandari s, atkin sl, kilpatrick es. the effect of iron and erythropoietin treatment on the a1c of patients with diabetes and chronic kidney disease. diabetes care. 2010 nov;33(11):2310–3. 13. alap l. christy, poornima a. manjrekar, ruby p. babu, anupama hegde, rukmini m.s. influence of iron deficiency anemia on hemoglobin a1c levels in diabetic individuals with controlled plasma glucose levels. iran biomed j. 2014 apr; 18(2): 88–93. 14. somiman a, de sanctis, yassin m, soliman n. iron deficiency anemia and glucose metabolism. acta biomed. 2017; 88(1): 112–118. 15. urrechaga e. influence of iron deficiency on hb a1c levels in type 2 diabetic patients. diabetes metab syndr. 2018 nov;12(6):1051-1055. 16. bhardwaj k, sharma sk, rajpal n, et al. effect of iron deficiency anaemia on haemoglobin a1c levels. ann clin lab res. 2016 october; 4: 4. 17. schindler c , birkenfeld al, hanefeld m, schatz u, köhler c, grüneberg m, tschöpe d, blüher m, hasslacher c, bornstein sr. intravenous ferric carboxymaltose in patients with type 2 diabetes mellitus and iron deficiency: clever trial study design and protocol. diabetes ther. 2018 feb;9(1):37-47. doi: 10.1007/s13300-017-0330-z. epub 2017 nov 13. 18. lavanya rajagopal, shivashekar ganapathy, sundaram arunachalam, veena raja, balaji ramraj. does iron deficiency anaemia and its severity influence hba1c level seemedj 2020, vol 4, no. 1 stigmatization of patients with viral infections and mass psychogenic illness 81 southeastern european medical journal, 2020; 4(1) in non diabetics? an analysis of 150 cases. j clin diagn res. 2017 feb; 11(2): ec13–ec15. 19. silva jf, pimentel al, camargo jl. effect of iron deficiency anaemia on hba1c levels is dependent on the degree of anaemia. clinical biochemistry. 2016;49(1):117–20. 20. el-agouza i, abu shahla a, sirdah m. the effect of iron deficiency anaemia on the levels of haemoglobin subtypes: possible consequences for clinical diagnosis. clin lab haematol. 2002;24(5):285–89. 21. bellamy l, casas jp, hingorani ad, williams d. type 2 diabetes mellitus after gestational diabetes: a systematic review and meta-analysis. lancet. 2009;373:1773–1779. 22. hashimoto k, noguchi s, morimoto y, hamada s, wasada k, imai s, et al. a1c but not serum glycated albumin is elevated in late pregnancy owing to iron deficiency. diabetes care. 2008 oct;31(10):1945–8. 23. hashimoto k, koga m. indicators of glycemic control in patients with gestational diabetes mellitus and pregnant women with diabetes mellitus. world j diabetes. 2015 jul 25; 6(8): 1045–1056. 24. vanheyningen c, dalton rg (1985) glycosylated haemoglobin in iron-deficiency anaemia. lancet 1: 874. 25. mitchell tr, anderson d, shepperd j (1980) iron deficiency, haemochromatosis, and glycosylated haemoglobin. lancet 2: 747. 26. solomon a, hussein m, negash m, ahmed a, bekele f, kahase d. effect of iron deficiency anemia on hba1c in diabetic patients at tikur anbessa specialized teaching hospital, addis ababa ethiopia. bmc hematol. 2019; 19: 2. 27. nitin sinha, m.d., t.k. mishra, m.d., tejinder singh, m.d., naresh gupta. effect of iron deficiency anemia on hemoglobin a1c levels. ann lab med. 2012 jan; 32(1): 17–22. 28. soliman a, de sanctis v, yassin m, soliman n. iron deficiency anemia and glucose metabolism. acta biomed. 2017; 88(1): 112–118. 29. koga m, murai j, morita s, saito h, kasayama s. comparison of annual variability in hba1c and glycated albumin in patients with type 1 vs. type 2 diabetes mellitus. j diabetes complications. 2013;27:211–213. 30. hirsch ib, brownlee m. beyond hemoglobin a1c--need for additional markers of risk for diabetic microvascular complications. jama. 2010;303:2291–2292. 31. lee j. alternative biomarkers for assessing glycemic control in diabetes: fructosamine, glycated albumin, and 1,5anhydroglucitol. ann pediatr endocrinol metab. 2015 jun; 20(2): 74–78. seemedj 2017, vol 1, no. 2 nurses’ attitudes toward nursing research 71 southeastern european medical journal, 2017; 1(2) nurses’ attitudes toward nursing research 1 ana kovačević1, nada prlić2, biljana matijašević3 1 clinical hospital center osijek, otorhinolaryngology and head and neck surgery clinic, osijek, croatia 2 faculty of medicine osijek, department of nursing, medical ethics and palliative medicine, josip juraj strossmayer university of osijek, osijek, croatia 3 private nursing home for elderly and disabled persons, makloševac, croatia corresponding author: ana kovačević, msc nursing – anakovacevic9@yahoo.com received: september 21, 2017; revised version accepted: november 24, 2017; published: november 24. 2017 keywords: attitudes towards research, nursing research, nurses (kadivnik m, muller a, milić vranješ i, šijanović s, wagner j. progins mutation of progesterone receptors and its role in premature birth – an overview. seemedj 2017; 1(2); 55-70) abstract aim: the aim of the study was to examine the attitudes of nurses towards research in nursing. methods: the study involved 202 respondents. the respondents were nurses employed at osijek clinical hospital. the boothe's attitudes on nursing research scale was used as an instrument of research – a modified version by bostrom, a. c. prior to statistical data processing, respondents were divided into two groups, considering the level of education: vocational nurses and bachelors of science in nursing. by age, respondents were divided into three groups: aged 20 to 35, 36 to 50 and 51 to 65. the differences between the observed groups were tested by the t-test and analysis of variance. results: there is a statistically significant difference in attitudes towards research given the level of education of the respondents (p = 0.015). bachelors of science in nursing have more positive attitudes towards research (�̅�=148.5) compared to vocational nurses (�̅�=141.1). a significant difference in attitudes towards nursing research was determined also with respect to the age of respondents (p = 0.002). younger nurses have a more positive attitude towards research in nursing (�̅� = 151.5) than middle-aged (�̅�=140.9) and senior-aged nurses (�̅�=140.1) do. there is also a significant difference in the level of motivation for continuing professional education given the respondents’ level of education (p = 0.019). conclusion: nurses show mildly positive attitudes towards nursing research. (kovačević a, prlić n, matijašević b. nurses’ attitudes toward nursing research. seemedj 2017; 1(2); 71-80) seemedj 2017, vol 1, no. 2 nurses’ attitudes toward nursing research 72 southeastern european medical journal, 2017; 1(2) introduction “nursing research is systematic inquiry designed to develop trustworthy evidence about issues of importance to the nursing profession” (1). nursing research, in the narrow sense, refers to “a scientific process that validates and refines existing knowledge and generates new knowledge that directly and indirectly influences clinical nursing practice” (2). scientific research in nursing dates back to florence nightingale. her data collection and analysis of factors affecting mortality and the illness of soldiers during the war resulted in changes in health care (3). more precisely, although f. nightingale recommended conducting clinical nursing research back in the mid-19th century, it was almost 100 years later that most nurses accepted her counsel. still, there is reason for optimism. the number and quality of research in nursing is growing today (4). with the development of nursing education in the republic of croatia, the number of research projects in the field of health care has increased. however, in comparison to western world countries, there is still not enough research in nursing in our country. the availability of nursing journals through online databases provides nurses in croatia with a detailed insight into what has been hitherto investigated in the area of health care, allowing a comparison of the research results with those conducted in the world and facilitating the provision of evidence-based health care. evidence-based clinical nursing practice represents a connection between nurses' personal experience and patients' value system on the one side and evidence on nursing and medical literature on the other (5). evidencebased practice has shown correlation with better patient outcomes, as patient care decisions are then backed up by scientific evidence (6). research is an important factor in establishing and maintaining high standards of health care (7). some of the limitations of scientific research application, both in general and in healthcare, are: moral or ethical problems, problems of complexity of human functioning, problems related to measurement and control issues (8). research shows that nurses find the following to represent main obstacles to research: lack of time, lack of interest and motivation, insufficient awareness of available research literature, insufficient authority to change the practice, lack of peer support, lack of support for the implementation of research findings in practice and insufficient understanding of the research process (6,9). some of the goals of nursing research are to promote evidence-based nursing practice, to secure the credibility of the nursing profession, to determine nursing practice responsibility, and to document the cost-effectiveness of nursing care (4). estabrooks et al. (10) have conducted an integrative review of literature and a metaanalysis of studies that deal with the impact of individual nursing personalities on research use. there were 20 studies involved in the metaanalysis, and 6 categories of potential individual characteristics were found, divided by the authors as follows: a) beliefs and attitudes, b) involvement in research activities, c) search for information, d) professional characteristics, e) education, and f) socio-economic factors. the authors concluded that all of these studies show methodological problems and it would be very difficult to suggest that any other individual characteristic, other than one’s attitudes toward research, affects the use of research as such. attitude is an acquired, relatively durable and stable organization of positive or negative emotions, evaluations and responses to an object. once formed, attitudes are resistant to change and are quite durable. nevertheless, they can change under the influence of changed circumstances and new experiences. as the social behavior of a human is related to their attitudes, there is a great interest in examining attitudes in order to explain and predict behaviors or to influence behaviors by means of planned attitude-changing procedures (11). seemedj 2017, vol 1, no. 2 nurses’ attitudes toward nursing research 73 southeastern european medical journal, 2017; 1(2) many studies have focused on the attitudes of nurses towards research, as well as on conditions that facilitate their implementation. in 1989, bostrom a. c. et al. conducted a survey on 720 bachelors of science in nursing (“bsns”) and vocational nurses. the results of this study showed that both groups perceived that there was support for research activities both among their peers and in the hospital in which they were employed. they expressed their personal interest in participating in research activities and they strongly believed that clinical nursing practice was a suitable source of research in nursing. lack of time was identified as a leading factor for their personal lack of involvement in research, as well as a lack of cost-effectiveness in terms of rewards or job promotions (9). the results of marsh and brown's research, conducted in 1992 on 144 nurses employed in a private hospital, showed that nurses had mildly positive attitudes towards research. the degree of education was the only respondents’ characteristic that was shown to be related to their attitudes (12). bjorkstrom and hamrin concluded in their research that nurses generally have positive attitudes towards research. they found some discrepancies between age groups (younger nurses have a more positive attitude towards research) and in terms of the level of education (nurses with higher levels of education have more positive attitudes towards research) (13). in 2007, hofmeister examined the attitudes of nurses towards research and found that they had slightly positive attitudes. she also stated that, as the level of nurses’ education increases, so do their positive attitudes towards research. she concluded that time, support and education are the main factors that could lead to more positive attitudes towards research (6). a study conducted in sweden on a sample of 1054 nurses was based on the attitudes of nurses towards research as well as their awareness and use of research findings. the results of the survey show that nurses generally have positive attitudes towards research (14). the main purpose of this study is to examine attitudes of nurses towards research in nursing. specific goals are focused on the following issues: 1) to investigate whether there is a difference in attitudes towards research in nursing with respect to the level of respondents’ education, 2) to examine whether there is a difference in attitudes towards research in nursing with regard to the respondents’ age; 3) to find out whether there is a difference in the level of motivation for continuing professional education given the level of respondents’ education, and 4) to examine whether there is a difference in the level of motivation for continuing professional education with regard to the respondents’ age. materials and methods a cross-sectional study was conducted (15). the research was conducted in the period from may to august 2016 at osijek clinical hospital. the study involved 202 respondents. the respondents were nurses employed at the clinics, institutes and departments of osijek clinical hospital. the research instrument used was boothe's attitudes on nursing research scale, modified version by bostrom a. c. (9), with prior permission to use. the original questionnaire has 46 questions. the question: “nursing research is more essential in the medical setting than in the psychiatric setting” was eliminated from the questionnaire because of inappropriate application in our health system. the scale contains 3 subscales: interest and environmental support, payoff and benefits, and barriers to conducting research. answers are given on the likert 5-degree scale starting from 1 (i completely disagree) to 5 (i completely agree). the questionnaire results range in total from 45 to 225. a larger number indicates more positive attitudes towards research, while a high score on negative particles reflects negative attitudes. the total score on the first subscale can range from 20 to 100, on the second subscale from 17 to 85, and on the third from 8 to 40. reliability coefficients for the whole questionnaire and for all three subscales have been calculated. chronbach alpha for all the particles (with five reversed points) is 0.904. for the first subscale it seemedj 2017, vol 1, no. 2 nurses’ attitudes toward nursing research 74 southeastern european medical journal, 2017; 1(2) is 0.869, for the second subscale it is 0.839, and for the third subscale it is 0.439. the hofmeister’s study produced similar reliability coefficients for the three subscales (interest and environmental support 0.879, payoff and benefits 0.844, and for the third subscale barriers to conducting research it was 0.571) (6). the sociodemographic characteristics of respondents were collected through 6 questions (gender, age, level of education, workplace, level of motivation for further professional education and the number of nursing journals commonly read by the respondents). prior to statistical data processing, the respondents were divided into two groups, with regard to the level of education: bsns and vocational nurses. according to age, respondents were divided into three groups: aged 20 to 35, 36 to 50 and 51 to 65. statistical analysis categorical data were represented by absolute and relative frequencies. numerical data were described by arithmetic mean and standard deviation. the differences between the observed groups (level of education, age) were tested by ttest and analysis of variance. after the analysis of variance as a post hoc analysis, scheffe test was used. the level of significance was set at α = 0.05. the analysis of the data obtained was done by spps for windows (version 15.0, spss inc., chicago, il, usa) (16). ethical principles prior to the research, the written consent of the commission for ethical and vocational issues of nurses at the clinical hospital osijek was obtained on 21st april 2016, as well as the written consent of the principal nurses of the clinics and the institutes where the research was conducted. the research was conducted in accordance with ethical principles and human rights in research. results the study involved 202 respondents, of whom 19 (9.4%) were male and 183 (90.6%) female. out of the total number of respondents, 98 (48.5%) were bsns, and 104 (51.5%) were vocational nurses. the largest number of respondents worked at hospital departments (wards), 134 (66.3%). the age of respondents ranged from 21 to 64 (mean age was 40.4; sd = 11.2). overall results on the boothe's attitudes on nursing research scale range from 89 to 198, with arithmetic mean of 144.7 (sd = 21.7). the results on the first subscale (interest and environmental support) in this study range from 34 to 90, with an arithmetic mean of 62.7 (sd = 11.8), and the possible range from 20 to 100. on the subscale payoff and benefits, the results range from 29 to 76, with an arithmetic mean of 55.1 (sd = 10.0), and the possible range of 17 to 85. the results on the third subscale (barriers to conducting research) range from 18 to 37, with an arithmetic mean of 26.9 (sd = 4.0), and the possible range from 8 to 40 (table 1). average scores of individual particles for the three subscales are shown in tables 2, 3 and 4. bsns have achieved a significantly higher total score on the boothe's attitudes on nursing research scale compared to vocational nurses (t-test, p = 0.015). a statistically significant difference between bsns and vocational nurses has been found on the second subscale (payoff and benefits). in this subscale, vocational nurses score an average result of 53.1 (sd = 10.7), while the bsns achieve the average score of 57.2 (sd = 8.7) (t-test, p = 0.003). no statistically significant differences have been found between the observed groups on the two remaining subscales. in order to determine differences in attitudes towards research according to the age of respondents, the subjects were divided into 3 age groups: 20 to 35, 36 to 50 and 51 to 65 years of age. the analysis of variance confirmed that there was a statistically significant difference between the three age groups of respondents on the boothe's attitudes on nursing research scale. a post hoc analysis (scheffe test) confirmed the statistically significant difference between the first and the second age groups (p = 0.010), and seemedj 2017, vol 1, no. 2 nurses’ attitudes toward nursing research 75 southeastern european medical journal, 2017; 1(2) between the first and third age groups (p = 0.012). table 1. the attitudes of nurses toward nursing research the boothe's attitudes on nursing research scale min max 𝒙 (sd)* interest and environmental support 34 90 62.7 (11.8) payoff and benefits 29 76 55.1 (10.0) barriers to conducting research 18 37 26.9 (4.0) total score 89 198 144.7 (21.7) * arithmetic mean (standard deviation) table 2. the average scores of individual particles for subscale interest and environmental support item interest and environmental support 𝒙 (sd)* 1. i would like to conduct research. 3.0 (1.3) 2. i would like to put research high on my list of priorities. 2.8 (1.2) 4. i believe my place of employment would provide me with ample assistance during the research process. 3.4 (1.1) 5. i believe my place of employment would provide me with ample consultive assistance for conducting research. 3.5 (1.1) 6. my supervisor would allow time in my daily assignment to conduct research. 3.3 (1.2) 8. i know what is expected of me when submitting my research proposal to the hospital nursing research committee. 3.1 (1.0) 11. i am familiar with selected statistical procedures used for the analysis of research findings. 2.9 (1.1) 12. i believe my job provides the time necessary to conduct research. 2.4 (1.1) 13. my colleagues (other professionals) would encourage me to conduct research. 3.3 (1.1) 14. my peers in nursing would encourage conducting research. 3.5 (1.0) 15. i believe my peers in nursing would assist in conducting research. 3.5 (1.0) 16. my job provides ongoing educational programs in order to conduct research. 2.9 (1.1) 18. i believe my working environment provides ample opportunity to conduct research. 3.3 (1.0) 19. i believe my place of employment has ample secretarial assistance for anyone wishing to conduct research. 2.9 (1.1) 20. i believe my place of employment has ample statistical assistance for anyone wishing to conduct research. 2.8 (1.1) 21. i believe my place of employment has ample assistance for anyone for the analysis of results and findings of the research that is conducted. 2.8 (1.1) 27. nursing research requires more from me than i am willing to give to my job. 3.0 (0.9) 34. time spent giving patient care is more important than time spent conducting research. 4.1 (1.0) 35. i am interested in conducting research. 2.9 (1.2) 44. nursing research should be initiated by nurse researchers. 3.4 (1.2) seemedj 2017, vol 1, no. 2 nurses’ attitudes toward nursing research 76 southeastern european medical journal, 2017; 1(2) the post hoc analysis did not find any difference between the second and third age groups (p = 0.977). regarding the level of motivation for continuing professional education in relation to the respondents’ level of education, the results show that there is a statistically significant difference between the examined groups (p = 0.019). a higher level of motivation is present in bsn’s. in the group of bsn’s, a total of 8 (8.2%) respondents reported high motivation for continuing their education, whereas in the group of vocational nurses a total of 5 (4.8%) respondents reported high motivation. also, in the group of bsns, 7 of the respondents (7.1%) were not motivated to continue their professional education, while in the group of vocational nurses 19 of them (18.3%) were not motivated at all. results on the level of motivation for continuing professional education in relation to the respondents’ age show that the three age table 3. the average scores of individual particles for subscale payoff and benefits item payoff and benefits 𝒙 (sd)* 3. nursing research is conducted because it allows nurses to be promoted. 3.5 (1.1) 9. the informed consent necessary for employee participation in research prevents me from conducting research in my work areas. 3.3 (1.0) 22. i would conduct research if i had the time. 3.1 (1.2) 23. i would conduct research if i knew how to write the proposal, conduct and analyze the results and findings. 3.0 (1.2) 24. research findings that are advantageous to good patient care can be implemented in my working environment. 3.4 (1.1) 25. nursing research is the means whereby the theoretical basis for nursing practice is derived. 3.3 (1.2) 26. members of the treatment team other than nurses should conduct research relative to patient care. 3.3 (1.0) 29. i would like to conduct a study of a problem in patient care. 3.1 (1.1) 30. i would conduct research if patient assignments were lightened. 2.8 (1.1) 31. nursing research should be initiated by nurses in the clinical area. 3.4 (1.2) 33. nurses would conduct more research if more funds were available for them to use for this purpose. 3.7 (1.0) 36. nurses receive praise from their peers and colleagues when they conduct research. 2.8 (1.1) 37. nurses would conduct research if they were provided time for research. 3.7 (0.9) 38. nurses would conduct research if relief time were given to conduct research. 3.8 (1.0) 40. i would do research if i knew more about it. 2.9 (1.2) 41. nurses are criticized too much by their peers when they conduct research. 2.9 (1.0) 43. i believe that i would conduct research if someone more knowledgeable would help me in the process. 3.2 (1.2) * arithmetic mean (standard deviation) seemedj 2017, vol 1, no. 2 nurses’ attitudes toward nursing research 77 southeastern european medical journal, 2017; 1(2) groups differ to a statistically significant extent in terms of the level of motivation for further education (p <0.001). although in all three age groups most of the respondents are partially motivated to continue their professional education, the highest level of motivation is present in younger nurses (aged 20 to 35). also, among the nurses of younger age, only 2 of them (2.6%) were not motivated to continue their education, while the number of unmotivated nurses aged 51 to 65 was 13 (25%). the results also demonstrated that bsns commonly read more nursing journals compared to vocational nurses. the difference between these two observed groups was statistically significant (p = 0.001). most of the respondents in all three age groups usually read one nursing journal. however, 31 (40.8%) respondents in the youngest age group and only 7 (13.5%) of them in the age group of 51 to 65 read no nursing journals whatsoever. discussion the results of our research have shown that nurses show mildly positive attitudes towards nursing research. since the minimum possible score on the boothe's attitudes on nursing research scale is 45, and the maximum is 225, the resulting mean of 144.7 (sd = 21.7) indicates slightly positive attitudes towards research. these results are in line with the findings of previous studies (6, 7, 12, 17, 18), which also confirm that nurses’ attitudes towards research are positive. as hofmeister points out, such a positive attitude of nurses towards research is the key indicator of evidence-based practice (6). analyzing the overall outcomes on some subscales of boothe's attitudes on nursing research scale also confirms the positive attitudes of nurses towards research. the analysis of the results on the three subscales is consistent with the findings of hofmeister (6) and bostrom (9), who also found that nurses perceive that research support exists among their peers and at the hospital; they find that research is important and useful for nursing practice and express their awareness about obstacles to conducting research. in further analysis of the nurses’ attitudes towards research, an examination of subjects’ responses in individual particles of each subscale was performed. the average scores in individual particles of interest and environmental support subscale show that the respondents agreed with the statement that time spent in patient care was more important than the time taken to conduct research. this is also the particle for which the respondents showed the table 4. the average scores of individual particles for subscale barriers to conducting research item barriers to conducting research 𝒙 (sd)* 7. the process of submission of the research proposal to the hospital nursing research committee is too detailed. 3.3 (0.8) 10. the informed consent necessary for patient participation prevents me from conducting research in my work areas. 3.2 (1.0) 17. i have the skills and knowledge necessary for me to conduct research. 3.3 (1.0) 28. nursing research should be conducted by nurses with a baccalaureate degree. 3.7 (0.9) 32. nursing research should be initiated by nurses in education. 3.4 (1.1) 39. nursing research should be conducted by nurses with a doctorate. 3.3 (1.3) 42. nursing research should be conducted by nurses with a master's degree. 3.3 (1.2) 45. patient participation in nursing research is difficult to obtain. 3.3 (1.0) * arithmetic mean (standard deviation) seemedj 2017, vol 1, no. 2 nurses’ attitudes toward nursing research 78 southeastern european medical journal, 2017; 1(2) highest agreement in the whole questionnaire. also, respondents expressed a somewhat higher degree of agreement with statements saying that they believed they would get the help needed to conduct the research at their workplace and that their associates would help. the highest disagreement was expressed with regard to the statement that they had enough time at work to carry out the research. a somewhat lower score has also been achieved in the particle that says research is high on the list of their priorities. it can be concluded from such results that nurses, despite their generally positive attitudes towards research, nevertheless give greater priority to patient care, rather than conducting research. it is therefore questionable if they understand the importance of implementing research findings in their practice and if they see the resulting benefit to patients. concerning the payoff and benefits subscale, it has been shown that respondents agree the most with claims that nurses would conduct research if they were given free time and if they had more money available for that purpose. they agreed the least with the statement that nurses get praise from their associates and colleagues when conducting research. such results on the payoff and benefits as well as interest and environmental support subscale support earlier research results (6, 9), which also demonstrated that the time available, as well as working environment support, are important factors associated with the nurses’ decision whether they will implement and apply research findings or not. average scores on particular particles of the barriers to conducting research subscale show the respondents’ highest agreement with the statement that the research should be conducted by the bsns, which means that nurses are aware, to a certain extent, of the importance of education for acquiring knowledge of research methods. further analysis of nurses’ attitudes towards research is focused on examining differences in attitudes with regard to the level of education and the age of the respondents. our results show that bsns achieve a significantly higher total score on the boothe's attitudes on nursing research scale compared to vocational nurses. this finding is in line with the findings of earlier research (7, 12, 17, 18), which also showed that the higher the level of education among respondents, the more positive the attitudes towards research. given that nurses who attain a higher level of education get more familiar with the research process during their formal education, such findings are not surprising. as marsh and brown (12) point out, nurses who attended research methodology courses, either participating in research or were researchers themselves, have more positive attitudes towards research. bostrom (9) finds some additional differences between bsns and vocational nurses, and points out that bsns claimed that conducting research was a desirable part of their nursing role and considered themselves better prepared for conducting research, while vocational nurses believed that research is also important for patient care, but they were not willing to put research before patient care. consequently, the level of education is undoubtedly linked to positive attitudes towards research. education should be a fundamental element for motivating and strengthening positive attitudes towards research (18). there was a statistically significant difference between the three age groups of respondents on the boothe's attitudes on nursing research scale. although the results of some studies (12, 17) have not confirmed the correlation between age and attitudes towards nursing research, the findings of this study show that younger respondents have more positive attitudes towards research, as opposed to middle-aged and senior respondents. fugleberg (19) also found that younger nurses achieve higher results on scales that measure involvement in research and attitudes towards research. this finding is also in line with the results of our research and one may say that nurses of younger age have more positive attitudes than nurses of middle and senior age. regarding the level of motivation for continuing professional education, relative to the respondents’ level of seemedj 2017, vol 1, no. 2 nurses’ attitudes toward nursing research 79 southeastern european medical journal, 2017; 1(2) education, most respondents from both groups were only partially motivated to continue their professional education. nevertheless, the results show that a higher level of motivation is present in bsns. the results of the present study have also shown that the three age groups of respondents differ to a statistically significant extent in terms of the level of motivation for further education. although most of them are partially motivated to continue education, in all three age groups, it can be concluded that the highest level of motivation is present in younger nurses. although our research has shown that nurses have slightly positive attitudes towards research in nursing, which is consistent with previous research on this topic, the results of this study cannot be generalized to the entire population of nurses. a topic which has not been covered by this research, and may certainly be useful to examine, is the link between motivation for continuing education in one’s profession in general and their motivation toward nursing research specifically. another interesting topic for future research could be the relationship between attitudes towards research and actual conducting of research. namely, it is well-known that the attitude itself does not necessarily result in change of behavior. the fact that nurses have a positive attitude does not necessarily mean that they will also practice research. we may say that identifying the attitudes of nurses to research is only the first step that should link future studies to other significant variables in order to effectively assess the acceptance of changes in nursing practice and the conducting of evidence-based health care. conclusion based on the research conducted for the purposes of this paper, it can be concluded that nurses show mildly positive attitudes towards research in nursing. there is a significant difference in attitudes towards research with regard to the level of education and age of the respondents. bsns have more positive attitudes towards research than vocational nurses do. younger nurses have more positive attitudes towards nursing research than it is the case with middle-aged and senior-aged nurses. there is a significant difference in the level of motivation for continuing education in one’s profession with regard to the level of education and age of the respondents. a higher level of motivation is present in the bsns. the highest level of motivation is present in nurses aged between 20 and 35. acknowledgements the author would like to thank all the institutions in which this survey was conducted, as well as all the respondents who participated in the research, for their helpfulness. the paper was presented as a master’s thesis at the faculty of medicine of j. j. strossmayer university in osijek on may 15, 2017. funding: no specific funding was received for this study. transparency declaration: competing interests: none to declare. references 1. polit df, beck ct. essentials of nursing research: appraising evidence for nursing practice. 7th ed. philadelphia: wolters kluwer/lippincott/williams & wilkins health; 2010. 2. burns n, grove sk, gray jr. understanding nursing research: building an evidencebased practice. missouri: elsevier/ saunders; 2015. 3. polit df, beck ct. nursing research: principles and methods. 7th ed. philadelphia: lippincott williams & wilkins; 2004. 4. nieswiadomy rm. foundation of nursing research. 6th ed. boston: pearson; 2012. 5. ljubičić m, šare s. povezanost teorije i prakse u zdravstvenoj njezi. sg/nj 2015;20:254-6. 6. hofmeister n. attitudes of nurses toward research. master theses. grand valley state university; 2007. seemedj 2017, vol 1, no. 2 nurses’ attitudes toward nursing research 80 southeastern european medical journal, 2017; 1(2) 7. vijayalakshmi p, pashupu dr, nagarajaiah, thimmaiah r, math sb. nurses attitudes and perceptions of nursing research: an indian perspective. j nurs educ 2014;4(4):509-513. 8. kostović srzentić m, lučanin d. petrak o. nastavni tekstovi iz metodologije. katedra za zdravstvenu psihologiju. zdravstveno veleučilište u zagrebu. zagreb:2005. 9. bostrom ac, malnight m, macdougall j, hargis d. staff nurses’ attitudes toward nursing research: a descriptive survey. j adv nurs 1989;14:915-922. 10. estabrooks ca, floyd ja, scott – findlay s, o'leary k, gushta m. individual determinants of reserach utilization: a systematic review. j adv nurs 2003;43(5):506-520. 11. petz b, ed. psihologijski rječnik. jastrebarsko: naklada slap; 2005. 12. marsh gw, brown tl. the measurement of nurses’ attitudes towards nursing research and the research environment in clinical settings. j clin nurs 1992;1:315-322. 13. björkström em, hamrin kf e. swedish nurses’ attitudes towards research and development within nursing. methodological issues in nursing research. j adv nurs 2001;34(5):706-714. 14. nilsson kajermo k, alinaghizadeh h, falk u, wändell p, törnkvist l. psychometric evaluation of a questionnaire and primary healthcare nurses’ attitudes towards research and use of research findings. scand j caring sci 2013; 1-13. 15. marušić m. et al. uvod u znanstveni rad u medicini. 5th ed. zagreb: medicinska naklada; 2013. 16. petz b. osnovne statističke metode za nematematičare. 5th ed. jastrebarsko: naklada slap; 2004. 17. smirnoff m, ramirez m, kooplimae l, gibney m, dee mcevoy m. nurses’ attitudes toward nursing research at a metropolitan medical center. appl nurs res 2007;20:24-31. 18. mehrdad n, salsali m, kazemnejad a. iranian nurses’ attitudes toward research utilisation. j res nurs 2008;13(1):53-65. 19. wadman w. staff nurses’ attitudes and perceptions toward nursing research. memorial university of newfoundland. (thesis) 1997. http://collections.mun.ca/pdfs/theses/w adman_wanda.pdf seemedj 2018, vol 2, no. 1 the croatian translation of the horne and östberg morningness-eveningness… 1 southeastern european medical journal, 2018; 2(1) the croatian translation of the horne and östberg morningnesseveningness questionnaire with a brief review of circadian typology 1 jakov milić1*, ivana škrlec1, iva milić vranješ1,2, matea matić1, dubravka sertić3, marija heffer1 1 faculty of medicine osijek, josip juraj strossmayer university of osijek, croatia 2 clinic for gynecology and obstetrics, osijek university hospital, croatia 3 lipik elementary school, školska ulica 25, 34551 lipik, croatia corresponding author: jakov milić milic.jakov@gmail.com introduction human circadian rhythms are a result of an interaction of several factors, both external, such as light and temperature (1), and internal, determined by a circadian clock network consisting of molecular components where arntl, clock, crys and pers genes received: august 31, 2017; revised version accepted: april 4, 2018; published: november 27. 2018 keywords: circadian rhythms; daily rhythms; morningness; translation; chronobiology represent central nodes in the network (2, 3). the circadian system has a hierarchical structure. the suprachiasmatic nucleus (scn) of the hypothalamus is the ‘master clock’ and controls the activity of the peripheral clocks (4). two feedback loops, arntl/clock and cry/per control expression of downstream transcription factors which regulate downstream target abstract introduction: horne and östberg morningness-eveningness questionnaire (meq) is a questionnaire widely used to assess the circadian typology. the aim of this study was to translate the meq from english into croatian. methods: the translation process included two independent forward translations, integration of the forward translation into a single translation, back-translation, back translation review and drafting of the final translation. results: no semantic differences were observed when comparing the original and the backtranslation; thus, only minimal alterations were done to the final translation, compared to the first one. conclusions: the croatian version of the meq is a reliable translation ready to be tested in a croatian sample. (milić j, škrlec i, milić vranješ i, matić m, sertić d, heffer m. the croatian translation of the horne and östberg morningness-eveningness questionnaire with a brief review of circadian typology. seemedj 2018;2(1);1-11) seemedj 2018, vol 2, no. 1 the croatian translation of the horne and östberg morningness-eveningness… 2 southeastern european medical journal, 2018; 2(1) genes involved in different biochemical pathways (5). the circadian rhythm has been shown to have an impact on human metabolism and several medical conditions, such as diabetes, myocardial infarction, or stroke (6). different people tend to react differently to specific external factors, for example, with differences in the cyclic secretion of melatonin (7, 8), thus leading to differences in one's circadian rhythm. circadian typology shows important differences in biological and behavioral parameters and in circadian clock genes associated with sleepwake rhythm (9). the most common method of assessing human circadian preference is by using self-reported questionnaires in which the respondents choose the times in which they feel the best (10). the results of such questionnaires put the participants on a certain point of the morningness-eveningness (me) spectrum (11). on the one side of the spectrum there are the morning types. these individuals usually report early bedtimes and rise times, and they tend to perform better in the morning. on the other side of the spectrum there are the evening types, who report later bedtimes and rise times and tend to perform better later in the day (12). there are also intermediate or neither types, who fall somewhere in between the two extremes of the spectrum. based on the obtained scores in the morningness-eveningness questionnaire, individuals are classified into circadian typologies or chronotype (13). large epidemiologic studies have demonstrated that chronotype has a normal gaussian distribution that varies by age and gender, in that the young and old demonstrate earlier chronotype and adolescents and young adults demonstrate a later chronotype (1). several studies have confirmed the correlation of scores such as rmeq, mesc and similar, with objective measures such as the timing of participants’ body temperature, sleep-wake cycles, or cortisol or melatonin secretion rhythms (8, 14, 15). the genetics influences account for up to 50% of the variance in morningness, but other factors also have a significant influence, such as age and gender (16). the morningness-eveningness questionnaire has been used in a wide range of research contexts, including circadian rhythm sleep disorders and studies of genetic influences on sleep patterns (17, 18). one of the first validated, and still one of the most commonly used questionnaires is the horne and östberg morningness-eveningness questionnaire (meq). horne and östberg adapted and validated the questionnaire previously developed by öquist in 1970 (19). the questionnaire has since been translated into many different languages (19–21). to the authors’ knowledge, the translation made in the present study is the first croatian translation of this scale, which is significant when taking into account that this valuable instrument can be used for further research in this largely under-researched scientific area in the croatian population. the complete croatian version may be found in the supplement and can be freely used in other research. method morningness-eveningness questionnaire the questionnaire consists of 19 questions dealing with individual preferred times of activity, time of day in which the participants go to sleep or wake up, as well as alertness after waking up (10, 19). most answers are forcedchoice with no 'do not know/cannot decide' category. the summed scores can be used as a continuous variable, or they can be further divided into a five-point morningnesseveningness scale: definitely morning type (score 70-86), moderately morning type (score 59-69), neither type (score 42-58), moderately evening type (score 31-41), and definitely evening type (score 16-30) (10). the meq showed good internal consistency in several validation studies (20, 22). procedure the translation was performed following the algorithm presented in figure 1. the algorithm was decided upon after taking into account seemedj 2018, vol 2, no. 1 the croatian translation of the horne and östberg morningness-eveningness… 3 southeastern european medical journal, 2018; 2(1) procedures suggested in several articles (23– 29). to simplify the data input of the filled in questionnaires, discrete item choices (multiple choice form ranging from a to e) have been substituted for continuous graphic scales (a visual scale in which the participants had to tick the appropriate hours) (30) in questions 17 and 18, with the scoring remaining identical to the original scale. the wording was also slightly altered, as suggested by urbán et al. (30). the questions in each stage of the translation procedure can be observed in tables 1 and 2. results the translation process can be viewed in tables 1 and 2. table 1 shows each question in the original, the translation obtained by combining the two independent forward translations, the back-translation into english, as well as the final version of the translation. table 2 shows the two independent forward translations into croatian. the translators aimed to provide a translation that is as close to the original as is possible, with no significant changes to the meaning, or style. when comparing the english version and the back-translated version, nosemantic differences were observed. the only differences between the original and the back translation were related to the use of different grammatical forms, which results in very similar meaning. the example of this may be seen in questions 1 and 2, where "considering only your own 'feeling best' rhythm" of the original was back-translated into "guided only by your own 'feeling best' rhythm". in question 11, the original construct "you wish to be at your peak performance" was back-translated into "you want to be at your best". several other constructs also had a different wording, but with no relevant changes in meaning. discussion the horne and östberg morningnesseveningness questionnaire (meq) is one of the most widely used instruments in the area of circadian typology. it is easy to use, and the results of the test can be easily comparable to results of similar studies. in this study, the authors created a translation done in several steps, aiming to provide a reliable translation that can be applied in further research in the croatian-speaking population. limitations of the study this study failed to provide a validation of the final croatian translation in a sample of croatianspeaking participants, which should be performed in further research. figure 1. the algorithm of the translation protocol applied in this study seemedj 2018, vol 2, no. 1 the croatian translation of the horne and östberg morningness-eveningness… 4 southeastern european medical journal, 2018; 2(1) table 1. steps for the translation of the morningness-eveningness questionnaire into the croatian language and the final croatian version original combined translation back-translation final version 1. considering only your own "feeling best" rhythm, at what time would you get up if you were entirely free to plan your day? kad biste se vodili samo svojim ritmom u kojem se najbolje osjećate, kada biste se ustali kada biste bili posve slobodni isplanirati svoj dan? guided only by your own “feeling best” rhythm, at what time would you get up if you were entirely free to plan your day? vođeni samo osobnim ritmom u kojem se vi najbolje osjećate, kada biste se ustali kada biste bili posve slobodni isplanirati svoj dan? 2. considering only your own "feeling best" rhythm, at what time would you go to bed if you were entirely free to plan your evening? kad biste se vodili samo svojim ritmom u kojem se najbolje osjećate, kada biste otišli spavati kada biste bili posve slobodni isplanirati svoju večer? guided only by your own “feeling best” rhythm, at what time would you go to sleep if you were entirely free to plan your evening? vođeni samo osobnim ritmom u kojem se vi najbolje osjećate, kada biste otišli spavati kada biste bili posve slobodni isplanirati svoju večer? 3. if there is a specific time at which you have to get up in the morning, to what extent are you dependent on being woken up by an alarm clock? ako postoji specifično vrijeme kada morate ujutro ustati, u kojoj mjeri ste ovisni o tome da vas probudi budilica? if there is a specific time at which you have to get up in the morning, to what extent are you dependent on being woken up by an alarm clock? ako postoji specifično vrijeme kada morate ujutro ustati, u kojoj mjeri ste ovisni o tome da vas probudi budilica? 4. assuming adequate environmental conditions, how easy do you find getting up in the mornings? ako se pretpostave adekvatni okolišni uvjeti, koliko vam se lako ujutro ustati? assuming that the environmental conditions are adequate, how easy do you find it to get up in the morning? ako se pretpostave adekvatni okolišni uvjeti, koliko vam se lako ujutro ustati? 5. how alert do you feel during the first half hour after having woken up in the mornings? koliko se budno osjećate u prvih pola sata nakon buđenja ujutro? how alert do you feel in the first half hour after having woken up in the morning? koliko se budno osjećate u prvih pola sata nakon buđenja ujutro? 6. how is your appetite during the first half-hour after having woken in the mornings? kakav vam je apetit u prvih pola sata nakon buđenja ujutro? how is your appetite in the first half hour after having woken up in the morning? kakav vam je apetit u prvih pola sata nakon buđenja ujutro? 7. during the first half-hour after having woken in the morning, how tired do you feel? koliko se umorno osjećate u prvih pola sata nakon buđenja ujutro? how tired do you feel in the first half hour after having woken up in the morning? koliko se umorno osjećate u prvih pola sata nakon buđenja ujutro? 8. when you have no commitments the next day, at what time do you go to bed compared to your usual bedtime? kad nemate nikakvih obveza sljedeći dan, koliko kasnije odlazite u krevet u usporedbi s vašim uobičajenim vremenom? when you have no commitments the next day, at what time do you go to bed, compared to your usual bedtime? kad nemate nikakvih obveza sljedeći dan, koliko kasnije odlazite u krevet u usporedbi s vašim uobičajenim vremenom? seemedj 2018, vol 2, no. 1 the croatian translation of the horne and östberg morningness-eveningness… 5 southeastern european medical journal, 2018; 2(1) 9. you have decided to engage in some physical exercise. a friend suggests that you do this one hour twice a week and the best time for him is between 7.0-8.0 am. bearing in mind nothing else but your own "feeling best" rhythm, how do you think you would perform? odlučili ste se baviti nekom fizičkom aktivnošću. prijatelj vam predlaže da se njome bavite po sat vremena dva puta tjedno, a za njega je najbolje vrijeme između 7 i 8 sati. imajući na umu isključivo vaš ritam u kojem se najbolje osjećate, što mislite kako biste to obavili? you have decided to do a certain physical activity. a friend suggests that you do this for one hour twice a week, and the best time for him is between 7-8 a.m. bearing in mind nothing else but your own ‘feeling best rhythm’, how do you think you would do? odlučili ste se baviti nekom fizičkom aktivnošću. prijatelj vam predlaže da se njome bavite po sat vremena dva puta tjedno, a za njega je najbolje vrijeme između 7 i 8 sati. imajući na umu isključivo vaš ritam u kojem se najbolje osjećate, što mislite kako biste to obavili? 10. at what time in the evening do you feel tired and as a result in need of sleep? u koje se doba večeri osjećate umorno i kao posljedicu toga osjećate potrebu za snom? at what time in the evening do you feel tired and, as a result, in need of sleep? u koje se doba večeri osjećate umorno i kao posljedicu toga osjećate potrebu za snom? 11. you wish to be at your peak performance for a test which you know is going to be mentally exhausting and lasting for two hours. you are entirely free to plan your day and considering only your own "feeling best" rhythm, which one of the four testing times would you choose? želite biti u najboljem stanju za test za koji znate da će biti mentalno iscrpljujuć i trajati dva sata. posve ste slobodni isplanirati svoj dan. isključivo s obzirom na vaš ritam u kojem se najbolje osjećate, koje biste vrijeme testiranja, jedno od četiri ponuđena, izabrali? you want to be at your best for a test that you know is going to be mentally exhausting and lasting two hours. you are completely free to plan your day. considering only your ‘feeling best rhythm’,which one of the four testing times would you choose? želite biti u najboljem stanju za test za koji znate da će biti mentalno iscrpljujuć i trajati dva sata. posve ste slobodni isplanirati svoj dan. isključivo s obzirom na vaš ritam u kojem se najbolje osjećate, koje biste od četiri ponuđena vremena testiranja izabrali? 12. if you went to bed at 11.0 pm at what level of tiredness would you be? kad biste pošli u krevet u 23 sata, na kojoj biste razini umora (pospanosti) bili? if you went to bed at 23h/11 p.m., at what level of tiredness (sleepiness) would you be? kad biste pošli u krevet u 23 sata, na kojoj biste razini umora (pospanosti) bili? 13. for some reason you have gone to bed several hours later than usual, but there is no need to get up at any particular time the next morning. which one of the following events are you most likely to experience? iz nekog ste razloga otišli u krevet nekoliko sati kasnije nego inače, ali nema razloga za ustajanjem u neko posebno vrijeme sljedećega jutra. koji ćete od ova četiri događaja najvjerojatnije iskusiti? for some reason, you have gone to bed several hours later than usual, but there is no reason to get up at a particular time the next morning. which one of the four following events are you most likely to experience? iz nekog ste razloga otišli u krevet nekoliko sati kasnije nego inače, ali nema razloga za ustajanjem u neko posebno vrijeme sljedećega jutra. koji ćete od ova četiri događaja najvjerojatnije iskusiti? 14. one night you have to remain awake between 4.00-6.00 am in order to carry out a night watch. jedne večeri morate ostati budni između 4 i 6 ujutro da biste obavili noćnu stražu. nemate obaveza one night you have to remain awake between 4-6 a.m. in order to carry out a night watch. you jedne večeri morate ostati budni između 4 i 6 ujutro da biste obavili noćnu stražu. nemate obaveza seemedj 2018, vol 2, no. 1 the croatian translation of the horne and östberg morningness-eveningness… 6 southeastern european medical journal, 2018; 2(1) you have no commitments the next day. which one of the following alternatives will suit you best? sljedeći dan. koja će vam od ponuđenih alternativa najbolje odgovarati? have no commitments the next day. which of the following alternatives would suit you best? sljedeći dan. koja će vam od ponuđenih alternativa najbolje odgovarati? 15. you have to do two hours of hard physical work. you are entirely free to plan your day and considering only your own "feeling best" rhythm which one of the following times would you choose? morate odraditi dva sata teškog fizičkog rada. posve ste slobodni isplanirati svoj dan. isključivo s obzirom na vaš ritam u kojem se najbolje osjećate, koji biste od ponuđenih termina izabrali? you have to do two hours of hard physical work. you are completely free to plan your day. considering only your ‘feeling best rhythm’, which of the following times would you choose? morate odraditi dva sata teškog fizičkog rada. posve ste slobodni isplanirati svoj dan. isključivo s obzirom na vaš ritam u kojem se najbolje osjećate, koji biste od ponuđenih vremena izabrali? 16. you have decided to engage in hard physical exercise. a friend suggests that you do this for one hour twice a week and the best time for him is between 10.0-11.0 pm. bearing in mind nothing else but your own "feeling best" rhythm how well do you think you would perform? odlučili ste se baviti teškom fizičkom vježbom. prijatelj vam predlaže da se njome bavite jedan sat dva puta tjedno, a za njega je najbolje vrijeme između 22 i 23 sata. isključivo s obzirom na vaš ritam u kojem se najbolje osjećate, što mislite kako biste to obavili? you have decided to engage in hard physical exercise. a friend suggests that you do this for one hour twice a week, and the best time for him is between 10-11 p.m. considering only your ‘feeling best rhythm’, how do you think you would perform this? odlučili ste se baviti teškom fizičkom vježbom. prijatelj vam predlaže da se njome bavite jedan sat dva puta tjedno, a za njega je najbolje vrijeme između 22 i 23 sata. isključivo s obzirom na vaš ritam u kojem se najbolje osjećate, što mislite kako biste to obavili? 17. suppose that you can choose your own work hours. assume that you worked a five hour day (including breaks) and that your job was interesting and paid by results. which five consecutive hours would you select? zamislite da sami možete birati svoje radne sate. pretpostavite da radite pet sati u danu (uključujući stanke), da vam je posao zanimljiv i plaćen po učinku. koje biste vrijeme izabrali za početak svog radnog vremena? imagine that you can choose your own work hours. assume that you work five hours a day (including breaks) and that your job is interesting and paid by the results. at what time would you choose to begin your workday? zamislite da sami možete birati svoje radne sate. pretpostavite da radite pet sati u danu (uključujući stanke), da vam je posao zanimljiv i plaćen po učinku. koje biste vrijeme izabrali za početak svog radnog vremena? 18. at what time of the day do you think that you reach your "feeling best" peak? u kojem dobu dana smatrate da se najbolje osjećate? at what time of the day do you think you feel your best? u koje doba dana smatrate da se najbolje osjećate? 19. one hears about "morning" and "evening" types of people. which one of these do you consider yourself to be? ako čujete za izraz „jutarnji“ i „večernji“ tipovi ljudi, kako biste sebe svrstali? if you ever heard of the terms ‘morning’ and ‘evening’ types of people, how would you categorize yourself? ako biste ikad čuli za izraze „jutarnji“ i „večernji“ tipovi ljudi, kako biste sebe svrstali? seemedj 2018, vol 2, no. 1 the croatian translation of the horne and östberg morningness-eveningness… 7 southeastern european medical journal, 2018; 2(1) table 2. the comparison of two forward translations into croatian from the english original forward translation 1 forward translation 2 1. kad biste se vodili samo svojim ritmom u kojem se najbolje osjećate, kada biste se ustali kada bi bili posve slobodni isplanirati svoj dan? kad biste se vodili samo svojim ritmom u kojem se najbolje osjećate, kada biste se ustali kada bi bili posve slobodni isplanirati svoj dan? 2. kad biste se vodili samo svojim ritmom, u koliko sati biste se probudili kad biste mogli potpuno samostalno planirati dan? kad biste se vodili samo svojim ritmom u kojem se najbolje osjećate, kada biste otišli spavati kada biste bili posve slobodni isplanirati svoju večer? 3. ako postoji točno vrijeme u koje se morate probuditi ujutro, koliko ste ovisni o alarmu budilice da vas probudi? ako postoji specifično vrijeme kada morate ujutro ustati, u kojoj ste mjeri ovisni o tome da vas probudi budilica? 4. ako se pretpostavi da je danas prosječan, uobičajen dan, koliko vam je jednostavno ustajanje ujutro? ako se pretpostave adekvatni okolišni uvjeti, koliko vam se lako ujutro ustati? 5. koliko se osjećate koncentrirano prvih pola sata nakon jutarnjeg buđenja? koliko se budno osjećate u prvih pola sata nakon buđenja ujutro? 6. kakav imate apetit tijekom prvih pola sata nakon jutarnjeg buđenja? kakav vam je apetit u prvih pola sata nakon buđenja ujutro? 7. koliko se umorno osjećate tijekom prvih pola sata budnosti? koliko se umorno osjećate u prvih pola sata nakon buđenja ujutro? 8. kad nemate nikakvih obveza sljedeći dan, koliko kasnije odlazite u krevet u usporedbi s vašim uobičajenim vremenom odlaska u krevet? kada sutradan nemate obaveza, kada odlazite u krevet u usporedbi sa uobičajenim odlaskom u krevet? 9. odlučili ste se uključiti u umjereno fizičko vježbanje. prijatelj vam predloži da vježbate jedan sat dva puta tjedno i najbolje vrijeme za to jest između 7:00 i 8:00 ujutro. ako razmatrate samo svoj ritam, koliko mislite da biste dobro vježbali? odlučili ste se baviti nekom fizičkom aktivnošću. prijatelj vam predlaže da se njome bavite po sat vremena dva puta tjedno u, a za njega je najbolje vrijeme između 7 i 8 sati. imajući na umu isključivo vaš ritam u kojem se najbolje osjećate, što mislite kako biste to obavili? 10. u koje se doba noći osjećate umorno i pospano? u koje se doba večeri osjećate umorno i kao posljedicu toga osjećate potrebu za snom? 11. želite biti u najboljoj formi za ispit za koji znate da će biti mentalno iscrpan i da će trajati dva sata. kad biste mogli slobodno planirati cijeli želite biti u najboljem stanju za test za koji znate da će biti mentalno iscrpljujuć i trajati dva sata. posve ste slobodni isplanirati svoj dan. seemedj 2018, vol 2, no. 1 the croatian translation of the horne and östberg morningness-eveningness… 8 southeastern european medical journal, 2018; 2(1) dan i vodeći se samo svojim ritmom, koje biste vrijeme pisanja ispita izabrali? isključivo s obzirom na vaš ritam u kojem se najbolje osjećate, koje biste vrijeme testiranja, jedno od četiri ponuđena, izabrali? 12. ako biste legli u 23:00, koliko biste bili umorni? kad biste pošli u krevet u 23 sata, na kojoj biste razini umora (pospanosti) bili? 13. zbog nekog ste razloga legli nekoliko sati kasnije nego inače, ali ne postoji razlog za buđenje u neko određeno vrijeme sljedećega dana. koji ćete od sljedećih događaja vjerojatno doživjeti? iz nekog ste razloga otišli u krevet nekoliko sati kasnije nego inače, ali nema razloga za ustajanje u neko posebno vrijeme sljedećega jutro. koji od ova četiri događaja ćete najvjerojatnije iskusiti? 14. jedne noći morate ostati budni između 4:00 i 6:00 da biste odradili noćnu stražu. nemate obaveza sljedeći dan. koja vas od sljedećih opcija najbolje opisuje? jedne večeri morate ostati budni između 4 i 6 ujutro da biste obavili noćnu stražu. nemate obaveza sljedeći dan. koja će vam od ponuđenih alternativa najbolje odgovarati? 15. morate odraditi dva sata teškog fizičkog rada. možete potpuno slobodno planirati svoj dan. uzimajući u obzir samo svoj ritam, koje biste vrijeme odabrali? morate odraditi dva sata teškog fizičkog rada. posve ste slobodni isplanirati svoj dan. isključivo s obzirom na vaš ritam u kojem se najbolje osjećate, koji biste od ponuđenih termina izabrali? 16. odlučili ste se uključiti u tešku fizičku aktivnost. prijatelj vam predloži da vježbate jedan sat dva puta tjedno i najbolje vrijeme za to jest između 10:00 i 11:00 ujutro. ako razmatrate samo svoj ritam, koliko mislite da biste dobro vježbali? odlučili ste se baviti teškom fizičkom vježbom. prijatelj vam predlaže da se njome bavite jedan sat dva puta tjedno, a za njega je najbolje vrijeme između 22 i 23 sata. isključivo s obzirom na vaš ritam u kojem se najbolje osjećate, što mislite kako biste to obavili? 17. pretpostavite da možete birati svoje radno vrijeme. zamislite da radite pet sati dnevno (uključujući pauze), da vam je posao zanimljiv i da se plaća po učinku. kojih biste pet uzastopnih sati odabrali? zamislite da sami možete birati svoje radne sate. pretpostavite da radite pet sati u danu (uključujući stanke), da vam je posao zanimljiv i plaćen po učinku. kojih biste pet uzastopnih sati izabrali? 18. u koje doba dana mislite da se osjećate najbolje? u kojem dobu dana smatrate da se najbolje osjećate? 19. ako čujete za izraz „jutarnji“ i „večernji“ tipovi ljudi, kako biste sebe svrstali? često se može čuti o „jutarnjim“ i „noćnim“ tipovima ljudi. što mislite, koji ste vi tip? seemedj 2018, vol 2, no. 1 the croatian translation of the horne and östberg morningness-eveningness… 9 southeastern european medical journal, 2018; 2(1) appendix 1. the final version of the croatian morningness-eveningness questionnaire morningness/eveningness questionnaire upute: 1. pažljivo pročitajte svako pitanje prije nego odgovorite na njega. 2. odgovorite na sva pitanja. 3. na pitanja odgovarajte numeričkim redom. 4. na svako bi pitanje trebalo odgovoriti neovisno o odgovorima na druga pitanja. ne vraćajte se unatrag i ne provjeravajte već dane odgovore. 5. većina pitanje ima nekoliko ponuđenih odgovora. kod svakoga pitanja križić stavite uz samo jedan odgovor. pojedina pitanja imaju skalu umjesto ponuđenih odgovora. stavite križić na prikladno mjesto na skali. 6. na svako pitanje odgovorite što je iskrenije moguće. vaši odgovori i rezultati držat će se u strogoj tajnosti. 7. slobodno ostavite komentare ispod svakoga pitanja na mjestu predviđenome za to. 1. vođeni samo osobnim ritmom u kojemu se najbolje osjećate, u koje biste se vrijeme ustali kada biste bili posve slobodni isplanirati svoj dan? a) 5:00 – 6:30 b) 6:30 – 7:45 c) 7:45 – 9:45 d) 9:45 – 11:00 e) 11:00 – 12:00 2. vođeni samo osobnim ritmom u kojemu se najbolje osjećate, u koje biste vrijeme otišli spavati kada biste bili posve slobodni isplanirati svoju večer? a) 20:00 – 21:00 b) 21:00 – 22:15 c) 22:15 00:30 d) 00:30 – 1:45 e) 1:45 – 3:00 3. ako postoji određeno vrijeme kada morate ustati ujutro, u kojoj ste mjeri ovisni o tome da vas budi budilica? a) nimalo ovisan/ovisna b) pomalo ovisan/ovisna c) poprilično ovisan/ovisna d) veoma ovisan/ovisna 4. pod pretpostavkom da su okolišni uvjeti odgovarajući, koliko vam se lako ujutro ustati? a) nimalo lako b) ne veoma lako c) poprilično lako d) veoma lako 5. koliko se ujutro budno osjećate unutar prvih pola sata nakon buđenja? a) nimalo budno b) pomalo budno c) poprilično budno d) veoma budno 6. kakav vam je ujutro apetit unutar prvih pola sata nakon buđenja? a) veoma slab b) poprilično slab c) poprilično dobar d) veoma dobar 7. koliko se ujutro umorno osjećate unutar prvih pola sata nakon buđenja? a) veoma umorno b) poprilično umorno c) poprilično osvježeno d) veoma osvježeno 8. kad nemate nikakvih obveza sljedeći dan, koliko kasnije odlazite u krevet u usporedbi s uobičajenim vremenom vašega odlaska na spavanje? a) rijetko ili nikad kasnije b) manje od jedan sat kasnije c) 1-2 sata kasnije d) više od dva sata kasnije 9. odlučili ste se baviti nekom fizičkom aktivnošću. prijatelj vam predlaže da se njome bavite po sat vremena dva puta tjedno, a za njega je najbolje vrijeme između 7 i 8 sati. imajući na umu isključivo vaš ritam u kojemu se najbolje osjećate, što mislite – kakva bi bila razina vaše izvedbe? a) dobro bih to obavio. b) relativno bih to dobro obavio. c) bilo bi mi naporno. d) bilo bi mi veoma naporno. 10. u koje se doba večeri osjećate umorno i kao posljedicu toga osjećate potrebu za snom? a) 20:00-21:00 b) 21:00-22:15 c) 22:15-00:45 d) 00:45-2:00 e) 2:00-3:00 11. želite biti u najboljemu stanju za test za koji znate da će biti mentalno zahtjevan i da će trajati dva sata. posve ste slobodni isplanirati svoj dan. uzevši u obzir isključivo ritam u kojemu se najbolje osjećate, koje biste od četiri ponuđena vremena testiranja izabrali? a) 8:00-10:00 b) 11:00-13:00 c) 15:00-17:00 seemedj 2018, vol 2, no. 1 the croatian translation of the horne and östberg morningness-eveningness… 10 southeastern european medical journal, 2018; 2(1) d) 19:00-21:00 12. kad biste pošli u krevet u 23 sata, na kojoj biste razini umora (pospanosti) bili? a) nimalo umoran/umorna b) pomalo umoran/umorna c) poprilično umoran/umorna d) veoma umoran/umorna 13. iz nekog ste razloga otišli u krevet nekoliko sati kasnije nego inače, ali nemate razloga ustati u neko posebno vrijeme sljedećega jutra. koji je od četiri navedena događaja najvjerojatniji u vašemu slučaju? a) probudit ću se u uobičajeno vrijeme i neću ponovno zaspati. b) probudit ću se u uobičajeno vrijeme i nakon toga zadrijemati. c) probudit ću se u uobičajeno vrijeme, ali ću ponovno zaspati. d) probudit ću se nakon uobičajenog vremena. 14. jedne večeri morate ostati budni između 4 i 6 ujutro da biste obavili noćnu stražu. nemate obaveza sljedeći dan. koja će vam od ponuđenih alternativa najbolje odgovarati? a) ne bih otišao/otišla u krevet dok straža ne bi bila gotova. b) odrijemao/odrijemala bih prije i spavao/spavala poslije. c) dobro bih se naspavao/naspavala prije i odrijemao/odrijemala poslije. d) spavao/spavala bih samo prije straže. 15. morate odraditi dva sata teškog fizičkog rada. posve ste slobodni isplanirati svoj dan. uzevši u obzir isključivo ritam u kojemu se najbolje osjećate, koje biste od ponuđenih vremena izabrali? a) 8:00 10:00 b) 11:00 13:00 c) 15:00 17:00 d) 19:00 21:00 16. odlučili ste se baviti teškom fizičkim treningom. prijatelj vam predlaže da se time bavite dva puta tjedno po jedan sat, a za njega je najbolje vrijeme između 22 i 23 sata. uzevši u obzir isključivo ritam u kojem se najbolje osjećate, što mislite – koliko kvalitetno biste to obavili? a) dobro bih to obavio. b) obavio bih to relativno dobro. c) bilo bi mi naporno. d) bilo bi mi veoma naporno. 17. zamislite da možete sami birati svoje radno vrijeme. pretpostavite da radite pet sati u danu (uključujući stanke), da vam je posao zanimljiv i plaćen po učinku. koje biste vrijeme izabrali za početak svog radnog vremena? a) 4:00 – 8:00 sati b) 8:00 – 9:00 sati c) 9:00 – 14:00 sati d) 14:00 – 17:00 sati e) 17:00 – 4:00 sata 18. u koje doba dana smatrate da se najbolje osjećate? a) 5:00 – 8:00 sati b) 8:00 – 10:00 sati c) 10:00 – 17:00 sati d) 17:00 – 22:00 sata e) 22:00 – 5:00 sati 19. moguće je čuti za „jutarnji“ i „večernji“ tip ljudi, u koji biste od njih svrstali sebe? a) zasigurno „jutarnji“ tip b) više sam „jutarnji“ nego „večernji“ tip. c) više sam „večernji“ nego „jutarnji“ tip. d) zasigurno sam „večernji“ tip. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. roenneberg t, kuehnle t, juda m, kantermann t, allebrandt k, gordijn m, et al. epidemiology of the human circadian clock. sleep med rev 2007;11(6):429–38. 2. osland tm, bjorvatn b, steen vm, pallesen s. association study of a variable-number tandem repeat polymorphism in the clock gene period3 and chronotype in norwegian university students. chronobiol int 2011;28(9):764–70. 3. corella d, asensio em, coltell o, sorlí j v., estruch r, martínez-gonzález má, et al. clock gene variation is associated with incidence of type-2 diabetes and cardiovascular diseases in type-2 diabetic subjects: dietary modulation in the predimed randomized trial. cardiovasc diabetol 2016;15(1):4. 4. muro a, gomà-i-freixanet m, adan a, cladellas r. circadian typology, age, and the alternative five-factor personality model in an adult women sample. chronobiol int 2011;28(8):690–6. 5. staels b. when the clock stops ticking, metabolic syndrome explodes. nat med 2006;12(1):54–5. 6. boden-albala b, roberts et, bazil c, moon y, elkind ms v, rundek t, et al. daytime sleepiness and risk of stroke and vascular disease: findings from the northern manhattan study (nomas). circ cardiovasc qual seemedj 2018, vol 2, no. 1 the croatian translation of the horne and östberg morningness-eveningness… 11 southeastern european medical journal, 2018; 2(1) outcomes 2012;5(4):500–7. 7. roenneberg t, merrow m. entrainment of the human circadian clock. cold spring harb symp quant biol 2007 jan;72:293–9. 8. duffy jf, dijk dj, hall ef, czeisler ca. relationship of endogenous circadian melatonin and temperature rhythms to selfreported preference for morning or evening activity in young and older people. j investig med 1999;47(3):141–50. 9. randler c, gomà-i-freixanet m, muro a, knauber c, adan a. do different circadian typology measures modulate their relationship with personality? 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12 southeastern european medical journal, 2018; 2(1) to biofilm or not to biofilm? valentina živković1, tomislav kurevija1, ivana haršanji drenjančević1,2, maja bogdan1,3, maja tomić paradžik1,4 jasminka talapko1, domagoj drenjančević1,2 1 faculty of medicine, josip juraj strossmayer university of osijek, osijek, croatia 2 university hospital center osijek, osijek, croatia 3 institute of public health osijek-baranja county, osijek, croatia 4 institute of public health brod-posavina county, slavonski brod, croatia corresponding author: domagoj drenjančević, md, phd domagoj@mefos.hr received: september 29, 2017; revised version accepted: april 3, 2018; published: november 27, 2018 keywords: biofilm, p. aeruginosa, s. aureus abstract aim: the goal of this research is to examine the biofilm forming ability of staphylococcus aureus and pseudomonas aeruginosa clinical isolates in different in vitro conditions using meuller-hinton and luria-bertani broths. material and methods: 30 strains of pseudomonas aeruginosa and 30 strains of staphylococcus aureus obtained from clinical specimens were used. after preparing the suspensions of bacteria inoculated on broths, they were set on microtiter plates and the biofilm production was measured using the spectrophotometric reader on 550 nm. strains were classified into four categories: nonproducing, weak producers, moderate and strong producers, based on the comparison of optical density of samples and negative control. results: both tested species successfully formed a biofilm in both broths (p<0.01). p. aeruginosa strains had a higher percentage of strong producers in both in vitro conditions, in comparison with s. aureus strains (3.3% vs 50%). nevertheless, there is no statistically significant difference in biofilm formation between the strains, regardless the used broths, and there is no statistically significant difference between the biofilm forming ability of both species observed separately regarding in vitro conditions either. conclusion: both species have an ability to produce biofilm, which likely contributes to the pathogenicity and virulence of these bacteria and also leads to a better understanding of their in vivo characteristics to cause infections related to biofilm. (zivkovic v, kurevija t, harsanji drenjancevic i, bogdan m, tomic paradzik m, talapko j, drenjančević, d. to biofilm or not to biofilm? seemedj 2018;2(1);12-19) mailto:domagoj@mefos.hr seemedj 2018, vol 2, no. 1 to biofilm or not to biofilm? 13 southeastern european medical journal, 2018; 2(1) introduction biofilm formation is one of the additional bacteria virulence factors which is still an interesting subject for numerous researches. biofilm infections are becoming a major health problem in chronic infections and implants. biofilm is a multicellular structure that protects bacteria from adverse environmental factors, making them highly resistant to different antibiotics. it also stores nutrients, which serve the bacteria to survive, protects them from phagocytosis, and secures survival in the host organism. resistance to disinfectants is a very important characteristic of biofilm because it prevents removing bacteria from the surface, enabling such microorganisms to permanently colonize the human organism with pathological consequences. biofilm should be considered as a mobile functional community with the features of a complete microorganism because, among other things, they have homeostasis, circulatory system, genetic material exchange and metabolic activity, which ensure their further development (1). in addition, biofilm-protected bacteria are capable to disperse individual bacterial cells and decomposing parts of biofilm into the surrounding tissues and circulation system. but most importantly, on the surfaces of medical devices or in the human body, biofilm is made by microorganisms with the ability to produce an extracellular polymeric substance. these polymeric substances have an ability to incorporate a large amount of water into their structure and become highly hydrated (2). these solid-liquid barriers between the surface and the aqueous environment allow the community of biofilms optimal conditions for the growth and survival of microorganisms. also, biofilm is formed exclusively by the cells that produce polysaccharides in sufficient quantity (3). several environmental and genetic signals control each step of biofilm development and dispersal. accumulation of signal molecules in the environment allows each bacterial cell to estimate cell density or the total number of bacteria at that time – the quorum detection or quorum sensing phenomenon. colonization of medical devices is proportionally increased by surface irregularity and microorganisms bond more rapidly to hydrophobic surfaces such as plastic, rather than hydrophilic ones. (2,4). the appearance of biofilm on implants and various surgical implantable devices causes chronic infections, rejection of implants, ineffectiveness of the embedded device, organ damage, and sometimes even lethal outcome for the patient. the aim of this research is to examine the biofilm forming ability of staphylococcus aureus and pseudomonas aeruginosa clinical isolates in different in vitro conditions using mueller-hinton and luria-bertani broths. material and methods sample preparation this study included 60 bacterial strains, 30 pseudomonas aeruginosa and 30 staphylococcus aureus strains, obtained from different clinical specimens from 2007 to 2015 and isolated in microbiological laboratories at university hospital center osijek, croatia and in the general hospital slavonski brod, croatia. all bacterial strains are part of the collection of microbial strains kept at the department of microbiology and parasitology, faculty of medicine, university of osijek. microorganisms were identified according to standard microbiological methods and biochemical tests to the species level (5). after the bacteria had been grown on the blood agar plate during 1824 hours incubation, two to three individual colonies of bacterial cultures were taken and inoculated into vials with 3 ml of mueller-hinton (mh) (becton dickinson and co., cockeysville md, usa) and luria-bertani (lb) (difco r luriabertani broth, becton dickinson, usa) broth. the suspensions were incubated in the thermostat at 37°c for another 18-24 hours. after incubation, the tubes were well mixed (vortexed) and 20 μl from each suspension was transferred into new tubes with 2 ml mh and lb broth, which yielded suspensions of approximately 5x105 cfu/ml concentrations. after the preparation, suspensions were planted on flat bottom seemedj 2018, vol 2, no. 1 to biofilm or not to biofilm? 14 southeastern european medical journal, 2018; 2(1) polyester microtiter plates (copan, brescia, italy). wells with 100 μl of uninoculated mh and lb were used as the negative control and the remaining wells had 50 μl mh or lb broths which were planted with 50 μl of the prepared suspensions. the biofilm-producing strain acinetobacter baumannii atcc 19606 was used as a positive control. the microtiter plate was incubated in the thermostat for 18-24 hours at 37°c. after the incubation, the broth was shaken out and wells were washed three times with distilled water. at the end of the experiment, coloring with 0.1% crystal violet and solubilization with 95% ethanol was done (6). all measurements were done in triplicate. quantification of biofilm the final step was a spectrophotometric measurement of biofilm production on an enzyme immunoassays plate reader (biorad 93200 pr3100 tsc microplate reader) at 550 nm. the optical density (od) values were measured in every well of the plate and they represent biofilm production. the final results were reported as the optical density cut-off value (odc), which was calculated as average od for each sample made in triplicate increased by three standard deviations of negative controls. the results were classified into the following categories: non-producers, weak, moderate and strong biofilm producers (6,7) according to the criteria presented in table 1. table 1. the criteria for evaluating biofilm production od = average optical density value of biofilm production in a single well; odc = limit value of biofilm production (at least some biofilm produced) statistical analysis the results were processed using the statistical software package spss 19.0 (ibm corp., armonk, ny, usa), and the data processing was carried out by checking normality distribution and calculation of descriptive data, including the frequencies, percentages, median and interquartile ranges. wilcoxon test of equivalent pairs, χ2 test with fisher's exact test and cramer's v (φ) coefficient were utilized for the statistical significance testing of the differences between two or more independent groups. results the biofilm production ability data for both bacterial species regarding the in vitro nutrient condition (incubation in mueller-hinton and luria-bertani broths) are shown in table 2. data are presented as the average of triplicate measurement of optical density and includes medians and interquartile ranges for each variable used. 𝑶𝑫 < 𝑶𝑫𝒄 non-producers 𝑶𝑫𝒄 < 𝑶𝑫 < 𝟐 × 𝑶𝑫𝒄 weak producers 𝟐 × 𝑶𝑫𝒄 < 𝑶𝑫 < 𝟒 × 𝑶𝑫𝒄 moderate producers 𝟒 × 𝑶𝑫𝒄 < 𝑶𝑫 strong producers seemedj 2018, vol 2, no. 1 to biofilm or not to biofilm? 15 southeastern european medical journal, 2018; 2(1) table 2. the amount of biofilm formed, presented as the average optical density for s. aureus and p. aeruginosa using luria-bertani and mueller-hinton broths, in comparison to control staphylococcus aureus c q luria-bertani broth control 0.059 0.014 od (ar) 0.085 0.031 mueller-hinton broth control 0.076 0.011 od (ar) 0.097 0.043 pseudomonas aeruginosa luria-bertani broth control 0.032 0.017 od (ar) 0.318 0.481 mueller-hinton broth control 0.070 0.07 od (ar) 0.330 0.602 legend: c = median; q = interquartile range; od (ar) = average optical density by comparing the data for both bacteria and cultivation media (table 2), it can be seen that interquartile dispersal is greater for p. aeruginosa than for s. aureus strains. table 3. distribution of biofilm production in s. aureus and p. aeruginosa strains using luria-bertani and mueller-hinton broth (p<0.01, wilcoxon's equivalent pair test) staphylococcus aureus non-producers weak producers moderate producers strong producers f (%) f (%) f (%) f (%) lb 17 (56.7) 13 (43.3) 0 (0) 0 (0) mh 19 (63.4) 9 (30) 1 (3.3) 1 (3.3) pseudomonas aeruginosa non-producers weak producers moderate producers strong producers f (%) f (%) f (%) f (%) lb 0 (0) 8 (26.7) 4 (13.3) 18 (60) mh 4 (13.3) 6 (20) 5 (16.7) 15 (50) legend: lb = luria-bertani broth; mh = mueller-hinton broth; f = frequency it has been found that both bacterial species, s. aureus and p. aeruginosa successfully (to a statistically significant degree) created biofilm in both cultivation media (p<0.01, wilcoxon's equivalent pair test). seemedj 2018, vol 2, no. 1 to biofilm or not to biofilm? 16 southeastern european medical journal, 2018; 2(1) table 4. the contingency table for biofilm production of p. aeruginosa and s. aureus strains in luriabertani and mueller-hinton broth. pseudomonas aeruginosa luria-bertani broth staphylococcus aureus weak producers moderate producers strong producers total non-producers 2 2 13 17 6.7% 6.7% 43.3% 56.7% weak producers 6 2 5 13 20.0% 6.7% 16.7% 43.3% total 8 4 18 30 26.7% 13.3% 60.0% 100.0% pseudomonas aeruginosa mueller-hinton broth staphylococcus aureus nonproducers weak producers moderate producers strong producers total nonproducers 3 3 3 10 19 10% 10.0% 10.0% 33.3% 63.3% weak producers 0 2 2 5 9 0% 6.7% 6.7% 16.7% 30.0% moderate producers 0 0 0 0 0 0% 0% 0% 0% 0% strong producers 0 1 0 0 1 0% 3.3% 0% 0% 3.3% total 4 6 5 15 30 13.3% 20.0% 16.7% 50.0% 100.0% the correlation between the tested bacterial species according to their biofilm production ability is shown in table 4. there is no statistically significant difference in biofilm formation between s. aureus and p. aeruginosa strains in luria-bertani (fischer's exact test, p=0.075) or in mueller-hinton broth (fischer's exact test, p=0.359). the ability to produce biofilm depending on different cultivation conditions is shown in figures 1 and 2. staphylococcus aureus strains had very modest biofilm production in both broths: 43.3% of the strains seem to be weak producers and the remaining are non-producers in luria-bertani broth. there is even a smaller number of biofilm weak producers (30%) in mueller-hinton broth, and almost all remaining ones are biofilm non-producers, with the exception of one moderate (3.3%) and one strong (3.3%) producer. p. aeruginosa strains belong to strong biofilm producers in both in vitro conditions. in luria-bertani broth, all tested strains were shown as biofilm producers. weak producers accounted for 26.7% of the strains, moderate ones accounted for 13.3%, and 60.0% were strong producers. there was 20.0% of weak producers, 16.7% of moderate producers and 50.0% of strong producers in mueller-hinton broth, and 13.3% of the strains were biofilm nonproducers. there was no statistically significant difference between cultivation conditions and the ability to form biofilm either in s. aureus or in p. aeruginosa strains. seemedj 2018, vol 2, no. 1 to biofilm or not to biofilm? 17 southeastern european medical journal, 2018; 2(1) figure 1. biofilm production ability of s. aureus in luria-bertani and mueller-hinton broths (fischer's exact test, p=0.664). figure 2. biofilm production ability of p. aeruginosa in luria-bertani and mueller-hinton broths (fischer's exact test, p=0.476). discussion the main observation which arises from this study is that both bacterial species, grampositive s. aureus and gram-negative p. aeruginosa, successfully and to a statistically significant degree form biofilm in both tested broths (table 4). another important observation is that interquartile dispersal is greater in p. aeruginosa compared to s. aureus strains. p. aeruginosa strains have a higher incidence of extreme values and thus a greater range of results. however, no statistical difference was observed with regard to the medium in which biofilm production was measured. both species showed that biofilm production is more pronounced in luria-bertani medium by comparing the percentage, but no statistical significance has been established in statistical tests. although luria-bertani medium is a medium in which higher production is expected, other authors have also pointed out the possibility that biofilm production may be unexpectedly expressed depending on the conditions of bacterial growth. biofilm formation can be strongly affected both by growth media and by temperature (8,9). another study (10) has also shown that both of these bacterial species are biofilm producers, independently of the clinical specimen isolation origin (sputum, urine, urine catheter, etc.). in this study, which involved the application of congo agar and tube method, 0 5 10 15 20 non-producers weak moderate strong s. aureus luria bertani mueller hinton 0 5 10 15 20 non-producers weak moderate strong p. aeruginosa luria bertani mueller hinton seemedj 2018, vol 2, no. 1 to biofilm or not to biofilm? 18 southeastern european medical journal, 2018; 2(1) influence of the different in vitro conditions on biofilm forming ability of these two bacterial species was visible. (10) both of the bacterial species have been shown to be strong producers of biofilm, with more than 80% of strong producers found (10). in our study, p. aeruginosa strains were strong producers in 55% cases, equally in both broths, whereas s. aureus strains had only one strong producer (3.3%). by comparing the results of this small series of experiments, it is reasonable to assume that the biofilm forming ability is greatly influenced by cultivation conditions, that it is nutrient dependent and also has a significant role in antimicrobial susceptibility of biofilms. (11,12,13) also, it is very important to emphasize that the role of biofilm in the genesis of infections associated with medical devices is indisputable. microorganisms isolated from the samples of patients with these infections often exhibit the apparent ability to generate biofilms, as has been shown in many studies. (14,15) additionally, it is known that multiple bacterial species can cooperate and form complex networks with many defending mechanisms and built-in sophisticated protection against the human immune system and antimicrobials as well. (16) such polymicrobial biofilms are nowadays recognized as a significant factor in the pathogenesis of multiple infections in humans. conclusion the obtained results are in agreement with previous medical and microbiological knowledge of biofilm formation, which plays a pivotal role in numerous infections such as periodontitis, chronic prostatitis, bacterial vaginosis, chronic otitis media, osteomyelitis, chronic pulmonary infections in cystic fibrosis patients, and chronic wound infection, considering that the investigated bacterial species, s. aureus and p. aeruginosa, are the most common etiological pathogens of these infections. funding we greatly appreciate the financial support of university of osijek, faculty of medicine (research grant vif2016-mefos-27). transparency declaration competing interests: none to declare. references 1. milanov d, ašanin r, vidić b, krnjajić d, petrović j. biofilm – organizacija života bakterija u prirodnim ekosistemima. arhiv veterinarske medicine 2008;1:5-15. 2. rodney md. biofilms: microbial life on surfaces. journal list. 2002;8:881–90. 3. irie y, roberts ael, kragh kn, gordon vd, hutchison j, allen rj et al. the pseudomonas aeruginosa psl polysaccharide is a social but noncheatable trait in biofilms. mbio 2017;8:00374-17. 4. characklis wg, mcfeters ga, marshall kc. physiological ecology in biofilm systems. new york: john wiley & sons; 1990. p. 341–94. 5. jorgensen jh, pfaller ma, carroll kc, funke g, landry ml, richter ss, warnock dw. manual of clinical microbiology. washington, dc, usa: asm press, 2015. 6. milanov d, bugarski d, petrović j, rackov o. primena testa na mikrotitracionim pločama i mikroskopskih tehnika u ispitivanju sposobnosti nekih bakterijskih vrsta izolovanih od životinja da formiraju biofilm. arhiv veterinarske medicine 2010;3:23-37. 7. ghellai l, hassaine h, klouche n, khadir a, aissaoui n, nas f et al. detection of biofilm formation of a collection of fifty strains of staphylococcus aureus isolated in algeria at the university hospital of tlemcen. j bacteriol res 2014;6:1-6. 8. labrie j, pelletier-jacques g, deslandes v, ramjeet m, auger e, nash jh, et al. effects of growth conditions on biofilm formation by actinobacillus pleuropneumonia. vet res 2010;41:3. 9. nucleo e, steffanoni l, fugazza g, migliavacca r, giacobone e, navarra a et al. growth in glucose-based medium and exposure to subinhibitory concentrations of imipenem induce biofilm formation in a multidrug-resistant clinical isolate of seemedj 2018, vol 2, no. 1 to biofilm or not to biofilm? 19 southeastern european medical journal, 2018; 2(1) acinetobacter baumannii. bmc microbiol 2009;9:270. 10. rewatkar ar, wadher bj. staphylococcus aureus and pseudomonas aeruginosa biofilm formation methods. iosr-jpbs 2013;8:36-40. 11. rochex a, lebeault jm. effects of nutrients on biofilm formation and detachment of a pseudomonas putida strain isolated from a paper machine. water res 2007;41:2885-92. 12. henry-stanley mj, hess dj, wells cl. aminoglycoside inhibition of staphylococcus aureus biofilm formation is nutrient dependent. j med microbiol 2014;63:861-9. 13. bogdan m, drenjancevic d, harsanji drenjancevic i, bedenic b, zujic atalic v, talapko j et al. in vitro effect of subminimal inhibitory concentrations of antibiotics on the biofilm formation ability of acinetobacter baumannii clinical isolates. j chemother 2018;30:16-24. 14. pour nk, dusane dh, dhakephalkar pk, zamin fr, zinjarde ss, chopade ba. biofilm formation by acinetobacter baumannii strains isolated from urinary tract infection and urinary catheters. fems immunol med microbiol 2011;62:328-38. 15. rodríguez-baño j, martí s, soto s, fernándezcuenca f, cisneros jm, pachón j, et al. biofilm formation in acinetobacter baumannii: associated features and clinical implications. clin microbiol infect. 2008;14:276–8. 16. hotterbeekx a, kumar-singh s, goossens h, malhotra-kumar s. in vivo and in vitro interactions between pseudomonas aeruginosa and staphylococcus spp. front cell infect microbiol 2017;7:106. doi: 10.3389/fcimb.2017.00106. ecollection 2017. seemedj 2018, vol 2, no. 1 suicidality in depressive patients 20 southeastern european medical journal, 2018; 2(1) suicidality in depressive patients 3 andrijana mišković1, dunja degmečić1,2 1 faculty of medicine, josip juraj strossmayer university of osijek, university department of psychiatry, university hospital center osijek, osijek, croatia 2 department of psychiatry, clinical hospital centre osijek , osijek, croatia corresponding author: dunja degmečić, dunja.degmecic@mefos.hr received: july 12, 2018; revised version accepted: march 2, 2018; published: november 27. 2018 keywords: depressive disorders, suicidal ideation, hospitalization abstract aim: the aim of this research was to examine the incidence of suicides in patients with depressive disorders who were hospitalized at the psychiatric clinic of the clinical hospital centre osijek during 2015 and 2016, and to determine the relationship between suicidality and parameters measured in this research. methods: this research included 325 depressive patients hospitalized in the psychiatric clinic of the clinical hospital centre osijek during 2015 and 2016. data were collected from medical records of patients diagnosed with depressive disorders. to collect data, the authors used a questionnaire drafted for the purposes of this research requiring the following information to be filled in: age, gender, employment status, marital status, qualifications, number of children, the existence of suicide attempts or repeated suicide attempts, the way in which suicide was attempted, number of hospitalizations and treatment duration in years, number of suicide attempts, motivation for attempted suicide, psychiatric heredity, comorbidity. results: of the total number of respondents (n=325), 119 (36.6%) patients had suicidal behavior pattern in the past, significantly more in 2015 (fisher’s exact test, p<0.001), at present the suicidal behavior pattern had 134 patients, significantly more in 2015 (fisher’s exact test, p=0.04). eighty (24.6%) respondents had attempted suicide. thirty-three (41.3%) out of the 80 (24.6%) respondents who had attempted suicide were men and 47 (58.8%) were women. the existence of psychiatric heredity or attempted suicide in the family does not affect the suicide attempt of the respondents. conclusion: suicidal behavior patterns in depressive patients hospitalized in the psychiatric clinic of the clinical hospital centre osijek during 2015 and 2016. are common. (mišković a, degmečić d. suicidality in depressive patients. seemedj 2018; 2(1); 20-28) seemedj 2018, vol 2, no. 1 suicidality in depressive patients 21 southeastern european medical journal, 2018; 2(1) introduction according to available data from the registers of the croatian institute of public health, one can see that in 2013 there were more than forty thousand hospitalizations associated with mental disorders registered, while depressive disorders were the cause of more than 13% of hospitalizations and the cause of 11% of day hospital treatments used due to mental disorders. (1). according to the criminal code of the republic of croatia, suicide as a criminal act is prosecuted in the case when the suicide is encouraged by another person. the criminal processing is conducted exclusively on a person who encouraged another person to the attempts to commit suicide. (2) a number of risk factors that can lead to suicide and attempts to commit suicide have been identified, and. affective disorders or personality disorders are mostly addressed (3). there is a specific sequence of behavior which occurs before attempting suicide, i.e. the pre-suicidal behavior syndrome and appeal-phenomenon which are often neglected and are often ignored (4). ringel defined the phenomena of parasuicidal and presuicidal behavior, and thus gave an insight into the events of psychopathological changes that lead up to suicide attempts. the first phase, which ringel termed “insufficiency and narrowing”, is the period in which fear and sadness arise and are present to an extent which limits the individual in performing everyday activities. from such feelings, the second phase is developed, a phase of aggression, when an individual directs aggression towards him/ herself because of isolation from the environment. fantasy about suicide or the escape phase constitutes the third phase, which develops from a relief mechanism into concrete ideas by which the suicidal person seeks to escape to a better world. at that stage, he/she considers the techniques and methods of committing suicide and increasingly thinks of oneself as the late one. the fourth phase, the phase of mental anesthesia, is the period in which dissociation of the individual’s personality is noticed. one person performs the tasks of daily life, and the other is preparing for suicide (5). the rate of committing suicide in the general population is 20 : 1, while in clinical cases, in individuals with depressive episodes, the rate is much higher, ranging from 5-10 : 1(6). risk factors for committing suicide that most commonly occur in individuals suffering from depressive disorder are most commonly associated with symptoms of the disease or with specific situations, features of personality, the specifics of family medical history and similar. the groups affected by these disorders are melancholic depressive individuals with a high level of selfcriticism and low level of self-esteem, as well as adolescents and older adults (7). in the republic of croatia, in the period from 2000 to 2014, the suicide rate dropped from 20.9 to 16.3 per 100000 of citizens, but then the suicide rate grew to 722 (17 committed suicides per 100000 persons) in 2015. the ratio of men and women in the observed period ranges from 2.2 3.7 : 1. the rate of suicide in the general population grows with age, with the highest rates being found in the population older than 65. the most common way of committing suicide in both sexes within the general population is hanging (8). beside the risk factors, that there are also protective factors that reduce the possibility of suicide. this includes family and social support, pregnancy, postpartum period, a larger number of children in the family and strong religious beliefs. more protective factors are the care for health and regular physical check-ups, regular application of therapies and optimal physical activity (9). the aim of the present study is examine the incidence of suicides in patients with depressive disorders who were hospitalized at the psychiatric clinic of the clinical hospital centre osijek during 2015 and 2016, and to determine the relationship between sociality and parameters measured in this research. methods this research included 325 depressive patients hospitalized in the psychiatric clinic of the clinical hospital centre osijek during 2015 and 2016. data were collected from the medical records of patients diagnosed with depressive disorders. to collect data the authors used a questionnaire made for the purpose of this research with the following information: age, seemedj 2018, vol 2, no. 1 suicidality in depressive patients 22 southeastern european medical journal, 2018; 2(1) gender, employment status, marital status, qualifications, number of children, the existence of suicide attempts or repeated suicide attempts, motivation for attempted suicide, whether they live alone or with their families, psychiatric heredity, comorbidity. all data were collected from 15 january 2017 to 1 may 2017 and recorded so as not to reveal the identity of the patient.attempts, the way in which the suicide was attempted, number of hospitalizations and treatment duration in years, number of suicide. statistical analysis categorical data are presented with absolute and relative frequencies. numerical data are described by the median and the boundaries of the interquartile range. the differences in categorical variables were tested by the chisquared test and, if necessary, by fisher's exact test. the normality of distribution of numerical variables was tested by the shapiro-wilk test. the differences between the numerical variables between the two independent groups were tested by mann-whitney in the test (23.24). all p values are two-sided. the significance level (medcalc software bvba, ostend, belgium; http://www.medcalc.org; 2014) was used is set to alpha = 0.05. for statistical analysis, the medcalc statistical software version 14.12.0. results out of the total number of respondents (n=325), 119 (36.6%) patients exhibited a suicidal behavior pattern in the past, significantly more in 2015 than in 2016 (fisher’s exact test, p<0,001), at present the suicidal behavior was present at 134 patients, significantly more in 2015 than in 2016 (fisher’s exact test, p=0.04). eighty (24.6%) respondents had attempted suicide. from 80 (24.6%) respondents who had attempted suicide, 33 (41.3%) were men and 47 (58.8%) were women (table 1). table 1. suicidal behavior pattern in the past and in the present, suicide attempt according to the sex of respondents number (%) of respondents p* 2015 2016 total suicidal behavior pattern in the past yes 71 (47.3) 48 (27.4) 119 (36.6) < 0.001 no 79 (52.7) 127 (72.6) 206 (63.4) suicidal behavior pattern in the present yes 71 (47.3) 63 (36) 134 (41.2) 0.04 no 79 (52.7) 112 (64) 191 (58.8) suicide attempt yes 38 (25.3) 42 (24) 80 (24.6) 0.79 no 112 (74.7) 133 (76) 245 (75.4) total 150 (100) 175 (100) 325 (100) suicide attempt according to sex male 17 (44.7) 16 (38.1) 33 (41.3) 0.65 female 21 (55.3) 26 (61.9) 47 (58.8) total 38 (100) 42 (100) 80 (100) * fisher's exact test there were no significant differences in 2015 and 2016 in terms of the number of suicide attempts (table 2). seemedj 2018, vol 2, no. 1 suicidality in depressive patients 23 southeastern european medical journal, 2018; 2(1) table 2. number of suicide attempts in years 2015 and 2016 number of suicide attempts number (%) of respondents p* 2015 2016 total one 23 (60.5) 30 (71.4) 53 (66.3) 0.82 two 9 (23.7) 7 (16.7) 16 (20) three 1 (2.6) 1 (2.4) 2 (2.5) four 0 (0) 1 (2.4) 1 (1.3) five 1 (2.6) 1 (2.4) 2 (2.5) >five 4 (10.5) 2 (4.8) 6 (7.5) total 38 (100) 42 (100) 80 (100) * fisher's exact test place of residence, marital status, the basic diagnosis and treatment duration do not seem to be connected with the respondent’s attempted suicide. in terms of number of suicide attempts, there was a statistically significant difference between subjects who had had suicidal ideas in the past or who presently had such ideas on the one side and those who never had such ideas on the other, with the former subjects being the ones who attempted suicide more often (fisher’s exact test, p < 0.001) (table 3). table 3. respondents according to the basic diagnosis, suicidal behavior pattern and suicide attempt number (%) of respondents according to suicide attempts p* no yes total location rural 128 (52.2) 43 (53.8) 171 (52.6) 0.89 urban 117 (47.8) 37 (46.3) 154 (47.4) marital status married 159 (64.9) 48 (60) 207 (63.7) 0.39 single 42 (17.1) 11 (13.8) 53 (16.3) divorced 40 (16.3) 19 (23.8) 59 (18.2) in a relationship 4 (1.6) 2 (2.5) 6 (1.8) the basic diagnosis depressive episode (f32) 75 (30.6) 29 (36.3) 104 (32) 0.41 recurrent depressive disorder (f33) 170 (69.4) 51 (63.8) 221 (68) treatment duration 1 year 32 (13.1) 11 (13.8) 43 (13.2) 0.66 2 years 13 (5.3) 4 (5) 17 (5.2) 3 years 9 (3.7) 6 (7.5) 15 (4.6) 4 years 5 (2) 0 5 (1.5) 5 years 9 (3.7) 3 (3.8) 12 (3.7) >5 years 177 (72.2) 56 (70) 233 (71.7) suicidal ideas in the past yes 51 (20.8) 68 (85) 119 (36,6) < 0.001 no 194 (79.2) 12 (15) 206 (63.4) suicidal ideas in the present yes 74 (30.2) 60 (75) 134 (41.2) < 0.001 no 171 (69.8) 20 (25) 191 (58.8) total 245 (100) 80 (100) 325 (100) * fisher's exact test seemedj 2018, vol 2, no. 1 suicidality in depressive patients 24 southeastern european medical journal, 2018; 2(1) the median age of the respondents who tried to commit suicide was 53 years of age (interquartile range from 43 to 60 years). the age of respondents was from 17 to 76 years, similar to those who have not tried suicide (table 4). table 4. age of the respondents according to suicide attempt the median age (interquartile range) of the respondents who tried to commit suicide p* no yes total age of the respondents [years] 55 (49 62) 53 (43 60) 54 (46 61) 0.05 *mann whitney u test the median age of respondents with one single attempted suicide was 54 (interquartile range from 45 to 60 years old), while the median of respondents with more than one attempt was 51 (interquartile range from 37 to 62 years) without statistically significant differences between those two groups (table 5). table 5. age of the respondents with one single attempted suicide and respondents with more than one attempt the median age (interquartile range) of the respondents according to the number of suicide attempts p* one single attempted suicide more than one attempt total age of the respondents [years] 54 (45 60) 51 (37 62) 54 (46 61) 0.28 *mann whitney u test for 38 (11.7%) respondents, family problems were the motive to attempt suicide, interpersonal problems motivated 33 (10.2%) of the respondents, and there were 7 (2.2%) of the respondents without a clear motive. the existence of psychiatric heredity was present in 94 (28.9%) of the respondents, significantly more among respondents who attempt suicide in the 2015 than in 2016 (fisher’s exact test, p < 0.001), and 20 (6.2%) of the respondents had had experience with attempted suicide in their families (table 6). table 6. motive to attempt suicide, psychiatric heredity and attempted suicide in families in years 2015 and 2016 number (%) of respondents p* 2015 2016 total motive to attempt suicide family problems 17 (11.3) 21 (12) 38 (11.7) 0.87 interpersonal problems 19 (12.7) 14 (8) 33 (10.2) 0.20 without a clear motive 2 (1.3) 5 (2.9) 7 (2.2) 0.46 psychiatric heredity yes 59 (39.3) 35 (20) 94 (28.9) <0.001 no 84 (56) 138 (78.9) 222 (68.3) unknown 7 (4.7) 2 (1.1) 9 (2.8) total 150 (100) 175 (100) 325 (100) attempted suicide in family yes 11 (7.4) 9 (5.1) 20 (6.2) 0.49 no 137 (92.6) 166 (94.9) 303 (93.8) total 148 (100) 175 (100) 323 (100) * fisher's exact test seemedj 2018, vol 2, no. 1 suicidality in depressive patients 25 southeastern european medical journal, 2018; 2(1) the existence of psychiatric heredity or suicide attempts in their families does not affect the suicide attempts of the respondents themselves. the number of respondents who attempted suicide and who had positive psychiatric heredity was 53 (66.3%), while 169 (69%) of the respondents did not have any experience with attempting suicide in their families or positive psychiatric heredity (table 7). table 7. respondents according to psychiatric heredity and attempted suicide in the family number (%) of respondents according to suicide attempts p* no yes total psychiatric heredity yes 68 (27.8) 26 (32.5) 94 (28.9) 0.55 no 169 (69) 53 (66.3) 222 (68.3) unknown 8 (3.3) 1 (1.3) 9 (2.8) total 245 (100) 80 (100) 325 (100) attempted suicide in the family yes 16 (6.6) 4 (5.1) 20 (6.2) 0.79 no 228 (93.4) 75 (94.9) 303 (93.8) total 244 (100) 79 (100) 323 (100) * fisher's exact test discussion as the authors stated at the very beginning, affective diseases are most common among persons who commit suicide. the lifetime risk of suicide in patients with depression is 15% (10). it should be emphasized that in patients suffering from depressive disorders, the risk of suicide is twenty times higher than in the general population (11, 12). in present study, 9.2% of respondents indicated deliberate self-harm and suicide attempts as the reasons for their last hospitalization. on the other hand, data from literature show that, out of the total number of respondents, a quarter of them had attempted suicide either recently or in the past, without statistically significant differences according to the sex of the respondents. suicide is three times more common in men, which was not found within our selected patient population. the authors can see from the results that there are no statistically significant differences regarding sex of the person who committed suicide (13). in addition, from the literature authors could see that the ratio of suicide attempts in men and women varied based on sociodemographic characteristics, which indicates that in countries with a higher standard of living there is a higher frequency of suicide attempts in males, while the frequency of suicide attempts among women shows a higher percentage in countries of medium and low living standards (14). in present study, however, there was no statistically significant relationship found between the place of living, so we can conclude that, in the case of present population, the living standard according to the place of living did not have any influence on whether the respondent attempted suicide or not. the lowest rate of attempted suicides was found in married people seemedj 2018, vol 2, no. 1 suicidality in depressive patients 26 southeastern european medical journal, 2018; 2(1) and that single life increases the risk by 2 times. the same applies to divorced and widowed persons, whose risk is also twice as high as the risk found in singles (13). on the other hand, in present study there was no statistically significant relationship between the marital status of the patients or whether they lived alone or in a young society in his /her own family and the intention for committing suicide. published studies show that people suffering from depression with the intent to commit suicide are of the average age of 55 (15). in present population, the average age of respondents who had attempted suicide was 53 years of age, with no significant differences in relation to those who had not attempted suicide, which coincides with data from the literature and is included in their interquartile age range. in present study, we hypothesized that the number of hospitalizations is connected with the attempts of suicide, however, this was not shown to be statistically significant and corresponds to the data from literature, which indicates that the number of psychiatric consultations did not correlate with increased risk of suicide in depressed patients (16). the most common motives that we can find in literature are interpersonal relationships, lack of love in the family, and loss of control over their disease (17). present research has shown that the most common motives for suicidal behavior are family problems and interpersonal reasons. for example, kieholz's scheme of judgement and determination of suicidal tendencies has shown that the presence of suicide(s) in the patient’s immediate family or among other close relatives increases the risk of suicide possibility (18). we can notice how this scheme can be applied to our study because people with suicidal ideas in the past or those with suicidal ideas at the present time attempted suicide more often. numerous studies, including family studies, studies of twins and adoptive researches, associate family communication and the possibility of inheritance of suicidal behavior (reviewed by 19). it is mentioned that the risk for a depressive episode is an independently inherited factor (20). in present research, based on the selected population, the existence of psychiatric heredity or suicide attempts in the family did not affect the attempted suicide of the respondents. in the actual attempted suicide, the most common method of execution of the act involves deliberate self-harm by using drugs/intoxication, which is also the most persistent in the form of suicide attempts, with deliberately intoxication and the effects of alcohol being found much more often in 2015. deliberate self-harm by hanging, strangulation, and asphyxiation showed no significant differences during monitoring period. data from the world health organization show that the methods of attempted suicides are different in certain parts of the world. the authors can distinguish three most common methods of attempted suicides in the world: hanging, poisoning with organophosphates and pesticides, and suicides by firearms. given the fact that croatia is a part of europe, we have compared the obtained data with the rest of europe, where it has been shown that the most common methods of suicide attempts, in the respondents in this study, were deliberate selfharm by using drugs and suicide attempt with a firearm, which coincides with the results obtained in present study (21). summary of the findings of present study are: a) recurrent depressive disorder (f33) was more common in 2016, while depressive episode (f32) was more common in 2015 (out of the total number of patients suffering from a depressive disorder); b) the most common comorbidity diagnoses of the respondents were in the area of mental disorders and disorders of behavior, secondly there were diagnoses in the fields of circulatory system diseases, while the least comorbidity was found in the area of skin and subcutaneous tissue diseases; c) suicidal forms of behavior in terms of suicidal ideas in the past and suicidal ideas in the present were significantly more present in 2015; d) there were no significant statistical differences with regard to gender and age of the respondents in relation to their attempted suicides, e) place of residence, marital status, the basic diagnosis and treatment duration were not found to be connected with whether the respondent attempted suicide or not; e)subjects with suicidal ideas in the past or in the present seemedj 2018, vol 2, no. 1 suicidality in depressive patients 27 southeastern european medical journal, 2018; 2(1) attempted suicide more frequently, to a statistically significant degree, f) the most common motives of suicidal behavior patterns were family problems and interpersonal considerations; g) the existence of psychiatric heredity or attempts of suicide in the family were not found to be connected with suicide attempts of the respondents; h) the number of psychiatric hospitalizations was not found to be connected with suicide attempts of depressed patients. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. croatian institute of public health. department for mental disorders with psychoses registry and committed suicides registry. croatian committed suicide registry available from: http://www.hzjz.hr/sluzbaepidemiologija-zarazne-bolesti/odsjek-zamentalne-poremecaje-s-registrom-zapsihoze-i-registrom-izvrsenih-suicida/. date last accessed: 28 february 2017. 2. criminal code of the republic of croatia. criminal offences against life and limb. official gazette .2014;125/11, 144/12, 56/15, 61/15. 3. hawton k, van heeringen k. suicide. the lancet 2009;373:1372–1381. 4. folnegović-šmalc v, kocijan-hercigonja d, barac b. prevencija suicidalnosti. zagreb: multigraf; 2001. pg. 35-43. 5. ringel e. da odbaciš život?: refleksije o suicidu. zagreb: biblioteka ¨oko 3 ujutro¨; 1983. pg. 7-15. 6. mindoljević-drakulić a. suicid fenomenologija i psihodinamika. zagreb: medicinska naklada; 2013. pg. 100-103. 7. kozarić-kovačić d, jendričko t. suicidalnost i depresija. medicus 2004;1:77 – 87. 8. stevanović r, capak k, benjak t. croatian health statistics yearbook 2015. croatian committed suicides registry. croatian institute of public health 2016;289-90. 9. rihmer z. depression and suicidal behaviour. chichester:wiley-blackwell publishing. 2011. pg. 53-73. 10. guze sb, robins e. suicide and primary affective disorder. br j psychiatry 1970;117:437-8. 11. harris ec, barraclough b. suicide as an outcome for mental disorders, a metaanalysis. br j psychiatry 1997;170:205-28. 12. chen yw, dilsaver sc. lifetime rates of suicide attempts among subjects with bipolar and unipolar disorders relative to subjects with other axis i disorders. biol psychiatry 1996;39:896-9. 13. novak l, labura d. suicid u mladih i uloga prvostupnika sestrinstva u prevenciji suicida, final paper. zadar: university of zadar, department of health studies. 2016. 14. world health organization. preventing suicide: a global imperative. executive summary. available from: http://www.who.int/mental_health/suicid eprevention/exe_summary_english.pdf?ua= 1. date last accessed: 21 april 2017. 15. us department of health and human services. office of applied studies, substance abuse and mental health services administration (samhsa). suicidal thoughts, suicide attempts, major depressive episode, and substance use among adults. available from: http://www.samhsa.gov/data/2k6/suicid e/suicide.pdf. date last accessed: 21 april 2017. 16. roy a. depressed patients who suicide at their first attempt have had few admissions. depress anxiety 1999; 9:75-7. 17. marčinko d. teorija suicida. pro mente croatica 2003/2004;7:15—16. 18. folnegović-šmalc v, folnegović grošić p, henigsberg n, eds. farmakoterapija depresija. medicus 2004;1:31 – 39. 19. brent da, mann jj. family genetic studies, suicide, and suicidal behavior. am j med genet c semin med menet 2005;133(1):1324. seemedj 2018, vol 2, no. 1 suicidality in depressive patients 28 southeastern european medical journal, 2018; 2(1) 20. gershon es. genetics. new york: oxford university press; 1990. 373–401. 21. ajdacic-gross v, g weiss m, ring m, hepp u, bopp m, eds. methods of suicide: international suicide patterns derived from the who mortality database. available from: http://www.who.int/bulletin/volumes/86 /9/07-043489/en/. date last accessed: 21 april 2017. 22. silobrčić-radić m, jelavić m, tomić b, ćorić t,stevanović r, eds. mentalni poremećaji u republici hrvatskoj. zagreb: croatian institute of public health. 2011.33-34. 23. ivanković d. i sur. osnove statističke analize za medicinare. zagreb: medicinski fakultet sveučilišta u zagrebu; 1988. pg. 335-48 24. marušić m. i sur. uvod u znanstveni rad u medicini. 4. izd. udžbenik. zagreb: medicinska naklada; 2008. pg. 3290 seemedj 2018, vol 2, no. 1 suicidality in depressive patients 29 southeastern european medical journal, 2018; 2(1) seemedj 2017, vol 1, no. 1 evaluation of antibacterial activity of two different honeys… 67 southeastern european medical journal, vol 1, 2017. evaluation of antibacterial activity of two different honeys against clinical isolates of β-hemolytic streptococci group a 1 ivan kotris1, jasminka talapko2, domagoj drenjančević2,3 1 general hospital vukovar, department of internal medicine, vukovar, croatia 2 josip juraj strossmayer university of osijek, faculty of medicine, department of microbiology and parasitology, osijek, croatia 3 university hospital center osijek, clinical institute of transfusion medicine, osijek, croatia corresponding author: domagoj drenjančević, md, phd domagoj@mefos.hr received: march 15, 2017; revised version accepted: april 19, 2017; published: april 24. 2017 keywords: antibacterial activity, acacia honey, horse chestnut honey, beta-hemolytic streptococci group a abstract introduction: the aim of this investigation was to evaluate in vitro antibacterial activity of horse chestnut honey and acacia honey at different concentrations against clinical isolates of the β-hemolytic streptococci group a (bhs-a). honey’s active compounds have multiple therapeutic effects and it is used as traditional medicine for treatment and prevention of various illnesses. materials and methods: the antimicrobial effects of two honeys were tested on forty-four strains of bhs-a, isolated from the throat by a modified disk diffusion method. a bacterial suspension of bhs-a strains was plated onto müller-hinton agar with 5% defibrinated horse blood. using a sterile 8 mm diameter cork borer, wells were cut in the agar and into each was introduced 100µl of the different concentrations of the honey solution (25 %v/v, 50 %v/v, 75% v/v and 100%v/v). a disk of penicillin was added as the positive control. the plates were incubated aerobically for18-24 hours at 36 (± 1)°c and zones of inhibition were measured. results: the average diameter of the inhibition zones of acacia honey (100 %v/v) was 12.48 mm ± 1.73 mm, for acacia honey (75 %v/v) it was 11.06 mm±1.24 mm and for horse chestnut honey (100 %v/v) it was 11.08 mm ± 1.02 mm. the positive control showed the average diameter of 30.45 mm ± 3.21 mm. statistical significance (p < 0.05) was observed comparing the zone diameters of the acacia honey (100%) and antibiotic penicillin, and between the horse chestnut honey (100%) and antibiotic penicillin. conclusion: acacia and horse chestnut honeys exhibit limited but effective antibacterial activity upon clinical isolates of bhs-a. (kotris i, talapko j, drenjančević d. evaluation of antibacterial activity of two different honeys against clinical isolates of β-hemolytic streptococci group a. seemedj 2017;1(1);67-73) seemedj 2017, vol 1, no. 1 evaluation of antibacterial activity of two different honeys… 68 southeastern european medical journal, vol 1, 2017. introduction β-hemolytic streptococci group a (bhs-a) are the most common bacterial cause of tonsillopharyngitis. this microorganism can also cause acute otitis media, pneumonia, skin and soft-tissue infections; cardiovascular, musculoskeletal, and lymphatic infections, bacteremia and meningitis (1). classic streptococcal tonsillopharyngitis has an acute onset; produces concurrent dysphagia, headache, and upon examination is characterized by intense tonsillopharyngeal erythema, white-yellow exudates, and tender/enlarged palatine tonsils. patients with acute streptococcal tonsillopharyngitis should receive therapy with an antimicrobial agent in a dose and for a duration that is likely to eradicate the infecting organism from the pharynx. most oral antibiotic therapy must be administered for the conventional 10 days to achieve maximal rates of pharyngeal eradication of bhs-a (2-3). over the past decade, several research groups have focused their attention to the use of bee honey as a supplement therapy (4-6). the use of bee honey as a traditional remedy for microbial infections dates back to ancient times (7). aristotle, c.350 bc recommended that bee honey collected in specific regions and seasons (and therefore presumably from different floral sources) can be used for the treatment of different ailments (8). certain types of honey exhibit broad-spectrum antimicrobial activity and are effective against antibiotic resistant bacterial pathogens (9-13). the present study aimed to evaluate the antibacterial activity of two bee honey varieties (horse chestnut honey and acacia honey) onto forty-four clinical isolates of bhs-a. materials and methods bacterial strain forty-four clinical isolates of beta-hemolytic streptococci group a (bhs-a) were evaluated. all these clinical isolates were donated by the microbiology department of the public health institute of the county brodsko-posavska. isolates of bhs-a used in our study were collected from throat swabs and were positive to bhs-a. they were identified on the basis of zone diameter around a bacitracin disk (10 µg/disk) as well as on susceptibility testing and by a latex agglutination slide test for the grouping of streptococci of the lancefield groups a, b, c, d, f and g from culture plates as recommended by clsi (14). honey samples and dilution of honey two honey varieties were used: horse chestnut honey and acacia honey, which were purchased from the local market from local beekeepers. because of the intensified viscosity of acacia honey, different concentrations were made in sterile distilled water: 25 % v/v, 50 % v/v, 75 % v/v. this was done by dissolving the respective volumes: 8.3 ml, 16.6 ml and 25 ml of acacia honey, into corresponding volumes of sterile distilled water to give a 33.33 ml preparation. these were the tested different concentrations in addition to non-diluted horse chestnut honey, acacia honey and penicillin disk as the control. susceptibility testing of honey a bacterial suspension of bhs-a strains, equal to the mcfarland standard of 0.5 was prepared in saline. with sterile swabs (copan italia s.p.a, brescia, italy -plain swab sterile), the suspension was coated onto a müller-hinton agar with 5% defibrinated horse blood. using a sterile 8 mm diameter cork borer, five wells were cut in the agar and into each was introduced 100µl of the different concentrations of the honey solution. into the place of the sixth well, a disk of penicillin was added as the positive control. the plates were incubated aerobically for18-24 hours at 36 (± 1)°c and examined for zones of inhibition. the zones of inhibition were measured (in millimeters) and recorded. seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 69 southeastern european medical journal, vol 1, 2017. table 1. inhibition zones of acacia honey and horse chestnut honey against beta-hemolytic streptococci group a (bhs-a) isolates. beta-hemolytic streptococci group a isolates acacia honey (concentration of honey) horse chestnut honey 100% antibiotic penicillin 100% 75% v/v 50% v/v 25% v/v bhs-a1 12 11 0 0 10 28 bhs-a2 14 12 0 0 12 28 bhs-a3 11 10 0 0 10 30 bhs-a4 12 11 0 0 11 23 bhs-a5 16 14 11 0 12 25 bhs-a6 15 12 0 0 10 33 bhs-a7 13 10 0 0 11 33 bhs-a8 14 12 0 0 11 30 bhs-a9 15 12 9 0 12 33 bhs-a10 12 11 0 0 11 28 bhs-a11 13 9 0 0 0 28 bhs-a12 12 11 0 0 0 34 bhs-a13 12 12 0 0 11 31 bhs-a14 18 13 10 0 13 33 bhs-a15 12 11 0 0 11 32 bhs-a16 15 11 0 0 0 27 bhs-a17 15 14 0 0 12 31 bhs-a18 11 11 0 0 11 32 bhs-a19 0 0 0 0 0 25 bhs-a20 10 0 0 0 0 28 bhs-a21 12 10 0 0 0 26 bhs-a22 12 11 0 0 10 32 bhs-a23 12 12 0 0 10 29 bhs-a24 0 9 0 0 0 27 bhs-a25 11 0 0 0 11 31 seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 70 southeastern european medical journal, vol 1, 2017. statistical analysis statistical analysis was performed using the program sigmaplot version 12.0. the differences observed between the groups were tested by multiple comparison procedures (turkey test). the statistical significance level was confirmed at p < 0.05. results the activity of acacia honey and horse chestnut honey against bhs-a isolates and the inhibitions zones are shown in table 1. the average diameter of the inhibition zones of acacia honey (100 % v/v) on strains that were sensitive to the honey was 12.48 mm (standard deviation sd = 1.73 mm). the average diameter of the inhibition zones of acacia honey (75 % v/v) on strains that were sensitive to the honey was 11.06 mm (sd = 1.24mm). finally, the average diameter of the inhibition zones of horse chestnut honey (100 % v/v) on strains that were sensitive to the honey was 11.08 mm (sd = 1.02 mm). antibiotic penicillin showed the average diameter of the inhibition zones to be 30.45 mm (sd = 3.21mm). comparing the zone of inhibitions, by multiple comparison procedures, of different concentrations of bhs-a26 11 11 0 0 0 33 bhs-a27 11 0 0 0 0 28 bhs-a28 15 13 0 0 13 31 bhs-a29 11 0 0 0 10 32 bhs-a30 12 0 0 0 10 30 bhs-a31 11 10 0 0 0 32 bhs-a32 12 9 0 0 0 33 bhs-a33 13 10 0 0 0 26 bhs-a34 12 0 0 0 0 34 bhs-a35 12 10 0 0 0 32 bhs-a36 12 11 0 0 0 29 bhs-a37 13 11 0 0 0 40 bhs-a38 10 10 0 0 0 32 bhs-a39 10 0 0 0 0 33 bhs-a40 12 10 0 0 13 31 bhs-a41 11 10 0 0 10 28 bhs-a42 13 12 0 0 11 36 bhs-a43 11 11 0 0 0 30 bhs-a44 13 11 0 0 0 33 *the zones of inhibition were measured in millimeters seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 71 southeastern european medical journal, vol 1, 2017. honeys and penicillin (positive control), astatistically significant difference was observed with 100% acacia honey and100 % horse chestnut honey (p < 0.05). discussion honey has several properties that contribute to its antimicrobial activity. low ph and high osmolarity are combined with the enzymatic production of hydrogen peroxide that exerts an antimicrobial effect (15-16). hydrogen peroxide is the major contributor to the antimicrobial activity of honey (17). the different concentrations of this compound in different honeys result in their varying antimicrobial effects (7). in our research, we evaluated the antimicrobial activity of honey on bhs-a because, if in the case of positive antibacterial activity of honey on the bhs-a, it can be used primarily in the supportive therapy for e.g. tonsillopharyngitis. the use of honey is extremely uncomplicated and easily applicable. researchers who evaluated the antimicrobial effect of honey showed that certain microorganisms have better sensitivity to it. mittal et al. reported greater honey activity in vitro against staphylococcus aureus, and escherichia coli than on pseudomonas aeruginosa (18). cooper et al. reported honey activity on catalase negative, gram positive cocci (5); while hannan et al. reported honey activity on typhoidal salmonellae. when observing the antimicrobial activity of honey, the key element of effectiveness is the type of honey. certain types of honey, such as manuka honey, exhibit extensive antimicrobial activity. a group of researchers from ireland and australia reported that manuka honey has clinical potential and a greater antimicrobial effect in vitro against staphylococcus aureus, and escherichia coli than on pseudomonas aeruginosa (19, 20). a group of researchers from cameroon focused on the antimicrobial activity of honeys on clinical isolates of helicobacter pylori (6, 21, 22). the results of such research can provide alternative therapies against certain bacteria. in our research, it was observed that 100% of acacia honey and horse chestnut honey show maximum antimicrobial activity as shown in table 1. when the concentrations of honey were 50% v/v and 25% v/v there was not an antimicrobial effect. this conclusion of our study is inconsistent with the conclusion of other authors (6). it is assumed that such differences are due to the various kinds of honeys. in this study, in order to evaluate the antibacterial activity of two honey varieties (horse chestnut honey and acacia honey) upon clinical isolates of bhs-a, we concluded that honeys exhibit limited, but effective antibacterial activity. such results support the use of honey as a supplement therapy for bhs-a infections e.g. tonsillopharyngitis. further research is required to determine the in vivo activity of horse chestnut honey and acacia honey onto bhs-a. acknowledgements the authors would like to thank maja tomić paradžik msc, md, from the microbiology department of public health institute of the county brodsko-posavska, for donating clinical isolates of bhs-a strains. this research was presented as a poster/abstract at the 9th international scientific and professional conference with food to health, on the 13th of october, 2016 in osijek, croatia. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. pichichero megroup a beta-hemolytic streptococcal infections. pediatr rev 1998;19:291-302. 2. bisno al, gerber ma, gwaltney jm, kaplan el, schwartz rh. practice guidelines for the diagnosis and management of group a seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 72 southeastern european medical journal, vol 1, 2017. streptococcal pharyngitis. cid 2002; 35:113– 25. 3. regoli m, chiappini e, bonsignori f, galli l, de martino m.update on the management of acute pharyngitis in children. ital j pediatr 2011;37:10. 4. hannan a, barkaat m, usman m, gilani wa, sami w. in vitro antibacterial activity of honey against clinical isolates of multidrug resistant typhoidal salmonellae. pakistan j zool 2009;41:1-6. 5. cooper ra, molan le. testing the susceptibility to manuka honey of streptococci isolated from wound swabs. journal of apiproduct and apimedical science 2011;3:117-22. 6. nzeako bc, al-namaani f. the antibacterial activity of honey on helicobacter pylori. sultan qaboos univ med j 2006;6:71–6. 7. molan pc.the antibacterial activity of honey.bee world 1992;73:5-28. 8. molan pc. the antibacterial activity of honey: 2. variation in the potency of the antibacterial activity. bee world 1992;73:5976. 9. cooper ra, wigley p, burton nf. susceptibility of multiresistant strains of burkholderiacepacia to honey. lettappl microbiol 2000;31:20–24. 10. cooper ra, halas e, molan pc. the efficacy of honey in inhibiting strains of pseudomonas aeruginosa from infected burns. j burn care rehabil 2002;23:366–70. 11. cooper ra, molan pc, harding kg. the sensitivity to honey of gram-positive cocci of clinical significance isolated from wounds. j appl microbiol 2002;93:857–63. 12. blair se, cokcetin nn, harry ej, carter da. the unusual antibacterial activity of medical-grade leptospermum honey: antibacterial spectrum, resistance and transcriptome analysis. eur j clin microbiol infect dis 2009;28:1199–208. 13. french vm, cooper ra, molan pc. the antibacterial activity of honey against coagulase-negative staphylococci. j antimicrob chemother 2005;56:228–31. 14. cornaglia g, ligozzi m, mazzariol a, valentini m, orefici g, fontana r, et al.rapid increase of resistance to erythromycin and clindamycin in streptococcus pyogenes in italy 1993-1995. emerg infect dis 1996;2:33942. 15. white jw, subers mh, schepartz ai. the identification of inhibine, the antibacterial factor in honey, as hydrogen peroxide and its origin in a honey glucose-oxidase system. biochimbiophysacta 1963;73:57–70. 16. bang lm, buntting c, molan p. the effect of dilution on the rate of hydrogen peroxide production in honey and its implications for wound healing. j altern complement med 2003;9:267–73. 17. taormina pj, niemira ba, beuchat lr. inhibitory activity of honey against foodborne pathogens as influenced by the presence of hydrogen peroxide and level of antioxidant power. int j food microbiol 2001;69:217-25. 18. mittal l, kakkar p, verma a, dixit kk, mehrotra m. anti microbial activity of honey against various endodontic microorganisms-an in vitro study. j int dent med res 2012;1;9-13. 19. sherlock o, dolan a, athman r, power a, gethin g,cowman s, et al. comparison of the antimicrobial activity of ulmo honey from chile and manuka honey against methicillin-resistant staphylococcus aureus, escherichia coli and pseudomonas aeruginosa. bmc complement altern med 2010;10:47. 20. irish j, blair s, carter da. the antibacterial activity of honey derived from australian flora. plos one 2011;6:e18229. 21. ndip rn, malange takang ae, echakachi cm, malongue a, akoachere jt, et al.invitro antimicrobial activity of selected seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 73 southeastern european medical journal, vol 1, 2017. honeys on clinical isolates of helicobacter pylori. afr health sci 2007;7: 228–32. 22. manyi-loh ce, clarke am, ndip rn. detection of phytoconstituents in column fractions of n-hexane extract of goldcrest honey exhibiting anti-helicobacter pylori activity. arch med res 2012;43:197-204. seemedj 2018, vol 2, no. 2 treatment of giant cell arteritis 34 southeastern european medical journal, 2018; 2(2) review a proposed pathway for the treatment of giant cell arteritis: experience from a district general hospital in the united kingdom 1 mehdi raza 1, yasser el miedany 1 1 dartford and gravesham nhs trust corresponding author: mehdi raza, mehdiraza@nhs.net received: jan 29, 2019; revised version accepted: mar 13, 2019; published: mar 31, 2019 keywords: temporal arteritis, giant cell arteritis, glucocorticoids, temporal artery, biopsy abstract introduction: giant cell arteritis can be difficult to diagnose clinically. in those cases, where there is no certainty, there is more reliance on a temporal artery biopsy and radiological imaging to confirm the diagnosis. the purpose of this article was to identify the standard of care in individuals with suspected giant cell arteritis in a typical district general hospital and to offer a proposed pathway for treatment. methods: darent valley hospital has been managing giant cell arteritis for many years but there has always been a need for an outlined pathway to identify those at risk of cranial complications like visual loss to improve patient care. we evaluated the management of 70 individuals that had a temporal artery biopsy and followed their treatment journey. we extracted clinical specialist, emergency admission, operation theatre and histological data. we collected clinic follow up data over the following years to identify those that relapsed on treatment, stayed in remission or had complications. we propose a pathway to manage those individuals with giant cell arteritis in line with the new advances in treatment. results: ten patients were identified that had a histologically positive biopsy. reassuringly, most individuals with an obvious clinical diagnosis had high dose glucocorticoid treatment commenced before even being referred for a biopsy. nine individuals had visual ischemia out of which five lost their vision. conclusion: the presentation of a pathway will help streamline best medical and surgical practice and ensure the availability of urgent specialist treatment and to identify those at risk of ischemic complications. (raza m, el miedany y. a proposed pathway for the treatment of giant cell arteritis: experience from a district general hospital in the united kingdom. seemedj 2018; 2(2); 34-47) seemedj 2018, vol 2, no. 2 treatment of giant cell arteritis 35 southeastern european medical journal, 2018; 2(2) introduction presentation of giant cell arteritis temporal arteritis (ta) is a spectrum of signs and symptoms involving the neck and cranial arteries due to a chronic inflammatory disorder. this can cause headaches, tender scalp, jaw claudication and more importantly visual loss. a complication of giant cell arteritis (gca), it is a chronic granulomatous inflammation of medium to large sized arteries, which can involve the aorta, proximal upper limb, neck, and extra cranial arteries. irreversible blindness can occur if this arteritis involves the branches of the ophthalmic artery. it is called arteritic anterior ischemic optic neuropathy (aaion) if it involves the posterior ciliary branches and and/or the ophthalmic artery. it is important that the patient have high dose glucocorticoids commenced immediately to prevent the progression of visual loss. some individuals are at risk of losing their vision because of delay in commencing glucocorticoids (1). figure 1. clinical presentation of the patient with temporal arteritis (authors’ photograph) clinical presentation classically the patient will present to the clinic or to an emergency unit with headache and a tender scalp on palpation and would be unable to comb his/her hair. signs will be palpation of a prominent, thick and tender artery with or without a palpable pulse. fundoscopy might reveal papilledema on ophthalmological review. the erythrocyte sedimentation rate (esr), creactive protein (crp) and platelet count rise as an acute phase response. constitutional symptoms arterial lesions may be widespread and causes nonspecific symptoms like fever, malaise and night sweats. the fever can reach 38°c but fever of unknown origin is more common in gca. other features like malaise and weight loss may lead to the suspicion of malignancy leading to further investigations to rule out sinister causes. myalgia and muscle stiffness the proximal muscles involving the shoulder and pelvic girdle are involved but distal muscles can also be involved. muscle stiffness occurs commonly in the morning and can be debilitating and difficult to treat. muscles can be tender causing difficulty in examining the strength and tone. disuse leads to contractures and atrophy of muscles. the duration of symptoms can vary from weeks to months. joint symptoms patients can develop tenderness over joints especially over shoulder and hip joints. synovitis although uncommon, can occur in the knees, shoulders and wrists. vasculitis involving branches of the external carotid artery headache and scalp pain are 50-75 % of the presenting symptoms of gca. the headache is characteristically extra-cranial, boring and dull in nature. the occipital or posterior auricular branches can be involved leading to varied symptoms like difficulty in combing hair, pain behind the ear and inability to sleep using a pillow. jaw pain and claudication has been shown to be a common symptom in gca and can occur in almost 50% of patients (2). the involvement of lingual and maxillary arteries leads to pain in the buccinators, maxillary muscles and tongue on seemedj 2018, vol 2, no. 2 treatment of giant cell arteritis 36 southeastern european medical journal, 2018; 2(2) chewing and talking. there have been reports of tongue gangrene although uncommon but a serious effect of ischaemia due to disease in the lingual artery. there have been cases of scalp necrosis secondary to gca. this is caused by severe arteritis and occlusion of the branches of the superficial temporal artery (3). vasculitis involving branches of the ophthalmic artery in patients with gca, decreased vision secondary to arteritis is the most serious consequence. it appears that 40% of patients presenting with visual loss have headaches, constitutional symptoms and polymyalgia rheumatica (pmr). partial visual field loss can progress to permanent blindness eventually. if unilateral visual loss is not treated, the second eye can be involved and can go blind in 1-2 weeks (4). involvement of the central retinal artery is about 10%. retinal signs and symptoms like exudates, haemorrhage and vasculitis are seen. amaurosis fugax occurs in 10% of patients with gca. eighty percent of patients with amaurosis develop blindness and hence treating the patients urgently is of fundamental importance. branches of the ophthalmic artery also supply the muscles to the eye and can cause diplopia and ptosis (5). vasculitis of larger arteries the symptoms may be due to the involvement of the aortic arch and descending aorta. the incidence varies from 9-18%. thoracic aneurysms with the presence of giant cells in the arterial wall can occur after a decade of the successful treatment of gca. it typically presents in a younger age group and mostly in women. the majority of symptoms involve arm or lower limb claudication. subclavian steal syndrome can occur due to narrowing of aortic arch roots. aortic aneurysms and intestinal infarction can occur but renal vessel involvement is rare. gca and takayasu’s disease have overlapping signs, radiological findings and symptoms (6). polymyalgia rheumatica pmr syndrome can occur alone or in conjunction with giant cell arteritis. forty percent of patients with gca have pmr. characteristically it starts with aching in the shoulder and hip girdles and morning stiffness. muscle weakness and elevated muscle enzymes unrelated to pmr. the individual experiences intense fatigue and malaise. there is a raised erythrocyte sedimentation rate (esr) and c-reactive protein. it is a clinical diagnosis and is treated with low dose gc in contrast to gca where high dose gc is given. pmr can present with distal synovitis that can mimic rheumatoid arthritis. patients with pmr should be warned to report new headaches or visual symptoms that could be the hallmarks of incipient gca immediately (7). complications secondary to gca cranial 1. there can be a long interval between the onset of symptoms and the occurrence of blindness. temporal arteritis may not be apparent initially if the symptoms are subtle. most commonly acute anterior ischemic optic neuropathy develops (8, 9, 10). 2. vertigo, deafness and tinnitus. 3. the central or peripheral nervous system can be involved leading to strokes and transient ischemic attacks (9). 4. the extent of the involvement of the scalp vessels depriving one area of the scalp of an adequate blood supply can cause scalp necrosis. there have been studies describing scalp necrosis secondary to ischaemia. in some case reports there have been occasions where debridement and skin graft was required for the area of skin loss (3, 11). extracranial 1. intermittent claudication and rest pain of upper and lower extremities due to seemedj 2018, vol 2, no. 2 treatment of giant cell arteritis 37 southeastern european medical journal, 2018; 2(2) inflammation in more axial large vessels can lead to persistent symptoms despite gc treatment. this requires appropriate investigations like magnetic resonance imaging and colour doppler imaging. if symptoms persist, takayasu’s aortitis needs to be ruled out as it can remain subclinical and quiescent despite being on gc therapy (12, 13). 2. aortitis and aortic dissection needs to be high in the differentials if presenting with atypical symptoms, backache and renal failure due to dissection extending down to the renal vessels. 3. cardiac murmurs like aortic regurgitation can lead to heart failure. 4. individuals can present with constitutional and pmr like symptoms, fever of unknown origin and raised inflammatory markers with no obvious clinical evidence of any aortic branch involvement. incidence and epidemiology over a 50-year period in olmsted county, minnesota, there were 173 incident cases of gca during the 50-year study. of these, 79% were women. a cyclic pattern of annual incidence rates was apparent, with evidence of 6 peak periods the mean age at diagnosis was 74.8 years (14). there has been increasing interest in epidemiology of gca where it was found to be more common in certain families particularly in the scandinavian countries (15). investigations and diagnosis traditionally clinical suspicion from the usual unilateral throbbing headaches and a tender scalp lead to an esr test and an immediate prescription of high dose oral steroids as soon as possible. clinical suspicion is important with typical throbbing headaches, tenderness in the scalp and age over 50. tab (temporal artery biopsy) is still performed quite frequently for individuals with clinical findings of gca. tab has traditionally been the gold standard for a diagnosis (2, 16). in 1990 the american college of rheumatology after comparing 214 gca individuals and 593 patients with other forms of vasculitis, identified criteria to diagnose gca. for the traditional format classification, five criteria are: 1. age greater than or equal to 50 years at disease onset, 2. new headache localised to the temple, 3. temporal artery tenderness or decreased temporal artery pulse, 4. elevated erythrocyte sedimentation rate (westergren) greater than or equal to 50 mm/hour, 5. biopsy sample including an artery showing necrotizing arteritis (17). pathophysiology it is known that the inflammatory cascade activates early in the pathological process of gca. however, the initiating event (aetiology) remains uncertain. there are many suspected pathogens. endothelial injury leads to the cell mediated immunity to be stimulated leading to the activation of the antigen presenting cells to the endothelium. this triggers vascular dendritic cells and monocytes to produce interleukin-6 (il-6), il-1 and pentraxin 3 (18). this leads to the stimulation of the liver to produce creactive protein (crp). constitutional symptoms as if fever and myalgia develop that are very responsive to glucocorticoid therapy, which effectively inhibits production of cells stimulated by th17, which produces il-6. the arterial wall is infiltrated by th1 and th17 type 4p lymphocytes. th1 lymphocytes produce interferon-g (ifn-g) and which result in formation of multinucleated giant cells from interaction with macrophages. glucocorticoids have minimal effect on these cells showing why visual loss persists despite high dose applied (19). gc therapy can suppress mechanisms affected by the il-6 like th17 activity. the increasing number of circulating immune cells and the inability of dendritic cells to block access to the arterial wall via the pd-1 immune checkpoint suppress the natural defences. it is seemedj 2018, vol 2, no. 2 treatment of giant cell arteritis 38 southeastern european medical journal, 2018; 2(2) known that the notch 1 receptor on t cells and notch-jagged 1 ligand receptor on the vasavasorum in the endothelium allow permeability of t cells into the vessel wall (20). platelet derived growth factor (pdgf) is an important cytokine activating concentric intimal hyperplasia. pdgf comes from macrophages and giant cells differentiating it from other vasculopathies. the media of the vessel wall is the main site of injury. the medial macrophages secrete tissue destroying enzymes and cytokines like pdgf and vascular endothelial growth factor (vgef) that also initiate repair. this results in a hyperplastic intima leading to occlusion of the vessel lumen. the intima and media is the centre of the pathological process. cell adhesion molecules and endothelial cells play a pivotal role in the repair process and neovascularisation. cell adhesion molecules might regulate how leukocytes and endothelial cells interact differently in different temporal arteries (21). activated white cells and platelets cause vessel inflammation and vascular thrombo-embolic events, an important finding in diagnosing gca. the inflammatory changes are quite similar to polymyalgia rheumatica. however, pmr is more of a systemic disease. the symptoms are more constitutional like fever malaise and weight loss. more specifically, there is more proximal muscle pain and joint pain in the shoulder, neck and pelvis. there is considerable chance (50%) in patients of ta to have pmr. temporal artery biopsy procedure the individual is consented for the biopsy and then brought in to the operating theatre. ideally, duplex ultrasound helps in identifying the temporal artery. we infiltrate local anaesthetic under the skin. a sterile field is created using drapes. a transverse or longitudinal incision is made on the skin just over the artery. both ends of the artery are ligated with absorbable or nonabsorbable suture and a generous length is isolated. the artery is divided between the two ligatures. the skin is apposed after haemostasis with interrupted non-absorbable or continuous absorbable suture. a sterile dressing is applied to the wound. non-absorbable sutures on the face or temple are removed in 5 days in the community. there has been a suggestion that the intraoperative assessment can identify thickness, tortuosity, colour/pallor of the arterial wall, blood flow, and the lumen size. this gives enough evidence to confirm the macroscopic presence of ta and appropriate treatment can be commenced immediately. however, it depends on the experience of the surgeon. this was studied in 111 cases where the intraoperative findings were compared with the histological findings and showed a 100% negative predictive value for biopsy negative specimens (22). complications secondary to temporal artery biopsy are not common. there have been reports of skin necrosis after temporal artery biopsy due possibly to arteritis changes in smaller collateral arteries. facial nerve injury can occur due to inadvertently not picking a safe zone and injuring the main branches of the facial nerve. clinically this leads to weakness of muscles of facial expression and frontalis muscle weakness. four cases of facial nerve injury were identified in one study. this can result in a permanent eyebrow droop on one side. it has been advocated that biopsy be orientated at the more parietal than frontal region to avoid injury to the frontal branch of the facial nerve (23, 24, 25). histological assessment of the specimen the specimen is sent to the laboratory in a sterile pot with formalin. the dimensions of the specimen are measured especially the length. the specimen is bisected or trisected (3 mm each length) and then ‘embedded’. the specimen is dehydrated, cleared, and infiltrated with the embedding material. they are ready for external embedding. the tissue sample is placed into moulds along with liquid embedding material (such as agar, gelatine, or wax) which is then hardened. longer specimens need to have more sections taken. three levels each from the individual trisected specimen are examined after staining with haematoxylin and eosin. seemedj 2018, vol 2, no. 2 treatment of giant cell arteritis 39 southeastern european medical journal, 2018; 2(2) haematoxylin stains the nuclei blue and eosin stains the cytoplasm, red blood cells and collagen fibres. the remaining part of specimen is stained with elastic van giessen (evg) staining. this stain is useful in demonstrating atrophy of elastic tissue. specific immuno-histochemical stains are carried out in some institutes but the typical findings of giant cells and inflammatory cells (as below) help in earlier diagnosis (26). figure 2. focal granulomatous inflammation and sometimes giant cells are visible. courtesy library.med.utah.edu patients and methods study design this is a retrospective case series study of individuals that underwent temporal artery biopsy in a district general hospital darent valley hospital in kent. there is great interest in the sequence of events that eventually lead to a tab. here are reviewed the events and outcomes of individuals that had a temporal artery biopsy. to achieve an adequate number of cases, the audit covered a number of years from 2009 to the current year. case retrieval all data of individuals undergoing a temporal artery biopsy were retrieved from our theatre link of operation records on our intranet called ‘theatreman’. case notes identified by first identifying specific codes for ‘temporal artery biopsy’ from the information technology department and then were compared with operating theatre data and histological data. the case-notes were electronic through our electronic health records (ehr). biopsy results were extracted from the darent valley hospital online histopathology system. seventy-six patients had a temporal artery biopsy from 2009 to 2018. all biopsies carried out were direct referrals form the emergency department and clinics. we documented all correspondence from the general practitioner and specialists. each individual was evaluated using the american college of rheumatology (acr) criteria. the american college of rheumatology criteria were used to diagnose gca. scoring criteria included age, headache, scalp tenderness, esr and tab results. the biopsy confirmed the diagnosis despite many individuals scoring three or four based on clinical suspicion and esr. the biopsy criteria were the presence of inflammation and microscopic presence of chronic inflammatory cells in the muscle. there also is disruption of the internal elastic lamina with or without the presence of giant cells (27, 28). the history of glucocorticoids administration was assessed throughout the period of the individual’s illness. operation notes identified the date of the procedure and grade of surgeon. the time elapsed from surgical referral to biopsy were recorded in days. this data was then transferred on to an excel sheet held on a computer. data collection a standard audit questionnaire helped in retrieving data. a single surgeon collected all the data from the case notes and later transferred all the data on to a microsoft excel spreadsheet. the basic epidemiological data collected was age at biopsy, gender and mode of referral. presenting symptoms were unilateral or bilateral headache, visual symptoms and jaw claudication. signs recorded were esr, ophthalmologic examination signs and scalp tenderness. positive findings on scalp examination were tenderness by palpating the seemedj 2018, vol 2, no. 2 treatment of giant cell arteritis 40 southeastern european medical journal, 2018; 2(2) artery, thickening and pulsation/no pulsation. we observed the duration in days from the surgical referral to biopsy and the date of commencement of corticosteroids. we recorded age above 50 years and esr above 50 mm/sec as positive criteria according to the acr. the histology results documented were for all tabs whether positive or negative and the specimen length of the temporal artery. the grade of the operating surgeon was either a surgical consultant or registrar. we gave particular attention to the visual signs /symptoms like amaurosis fugax or blindness, the findings of fundoscopy and the ophthalmologist opinion. we documented the start date of steroid therapy and previous administration of steroids. the change in dose of steroids was not possible to evaluate throughout the patient’s history. figure 3. length of delay from diagnosis to biopsy date results there were 76 individuals in the series. only 10 temporal artery biopsy results were positive on histology for temporal arteritis. six patient’s notes were not retrievable. the presenting signs and symptoms were as per table 1. 0 20 40 60 80 100 120 140 0 10 20 30 40 50 60 70 d a y s number of cases seemedj 2018, vol 2, no. 2 treatment of giant cell arteritis 41 southeastern european medical journal, 2018; 2(2) table 1. the presenting signs and symptoms of the patients with temporal arteritis age > 50 years 64/70 esr > 50mm 19/70 temporal tenderness 49/70 temporal headaches 58/70 jaw claudication 12 acr scores acr score 1 acr score 2 acr score 3 acr score 4 acr score 5 6 16 32 11 2 time till surgery 1 week 2 weeks >2 weeks 17 8 55 biopsy length 1 cm or above <1cm no artery/inadequate sample 30 36 4 biopsy findings positive biopsy negative biopsy 10 60 (6 inadequate specimens, vein, too small) description of patients the mean age was 67.9. the youngest age to have a tab was only 38 and oldest was 87 years old. there were 48 women out of 70 cases (68% of the total). the mean erythrocyte sedimentation rate (esr) calculated was 34.9 from 66 documented individuals that had an esr done. the minimum esr documented was zero and maximum was 110. there were four patients with no record of an esr. headaches were the most common symptoms as observed in our study. the pain is typically sudden and localised to one side of the scalp. however, it can occur in the parietal, occipital and frontal regions as well. the headache is new and different from any other type of headaches. twelve patients never had headaches out of the 70 but still had a biopsy done. nine individuals with headaches had bilateral involvement. forty-nine of the 70 individuals had a tender scalp on examination as an emergency or in clinic review. some cases had documentation of thickness or non-pulsatile artery. nine individuals had pain in the muscles of mastication while eating and chewing characteristic of jaw claudication. the visual findings were amaurosis fugax, complete or partial visual loss or diplopia. five patients had complete visual loss. we recorded seemedj 2018, vol 2, no. 2 treatment of giant cell arteritis 42 southeastern european medical journal, 2018; 2(2) visual acuity testing and finding characteristic signs of acute anterior ischemic occlusive optic neuritis (aaion). four other patients had visual impairment with characteristic findings of aaion but managed to receive gc therapy urgently to avoid loss of vision. six individuals had vague symptoms like blurring of vision and diplopia. it is important to note that two individuals that had characteristic aaion did not have histologically positive biopsies. discussion the management of gca begins with the clinical diagnosis. a biopsy is occasionally required when there is no clear clinical diagnosis based on the acr criteria. the treatment revolves around the immediate treatment, prolonging remission and the prevention of relapse. the aim of immediate treatment is to control the immediate systemic inflammatory response and to avoid complications like acute visual loss. there have been extensive studies on the optimal treatment modalities for the last 20 years. glucocorticoid treatment remains the mainstay acute treatment for gca despite the risk of adverse events. the immediate side effects of steroids include glucocorticoid effects such as hyperglycaemia especially in patients with diabetes mellitus and mineralocorticoid effects such as fluid retention. other side effects include mania, psychosis and depression, osteoporosis, cushing’s disease, obesity, new onset diabetes mellitus, peptic ulcers and depression on reduction or cessation of steroids. less common effects are hepatic steatosis, avascular necrosis and weight gain. prolonged administration of prednisolone may lead to adrenal suppression. eventually, this may cause the body to temporarily lose the ability to manufacture natural corticosteroids especially cortisol, which results in dependence on prednisone. prednisone is tapered if taken for more than seven days. individuals with pmr are commonly on low dose prednisolone for chronic systemic symptoms. clinicians should be aware of the likelihood of needing higher doses of steroids to combat the effects of gca. meticulous evaluation based on the clinical signs and symptoms is compulsory to avoid injudicious use of high dose steroids in high-risk individuals. even in this era of evolving clinical practice where more and more individuals are performing duplex ultrasonography to confirm or rule out a clinical diagnosis of ta, a biopsy still carries importance. in our trust there is growing enthusiasm in performing dul in clinic on individuals with a suspicion of gca (29). previous studies have showed that between a quarter and one third of all patients having tab yield a positive temporal artery biopsy. in our case series, the results showed that 12% of biopsy specimens came back positive (30). of these positive biopsies, eight cases out of 10 were an acr score of three and above and probably did not need a biopsy. if we look at the clinical criteria, most patients had an acr score of 3-4 fulfilling the diagnosis of temporal arteritis on the first clinical encounter and at the most could have benefitted with dul without the need for a biopsy. we can safely treat patients based on clinical presentation and laboratory esr findings. this will avoid unnecessary use of steroids on individuals with low clinical suspicion of gca. we know that a biopsy will have a higher chance of being positive in the first two weeks than later. twenty-five biopsies were performed within 2 weeks in our series. narvaez et al in his study evaluated the effects of duration of steroid treatment on biopsy results. the highest positive results were achieved (78%) in the individuals that had a biopsy within 2 weeks of commencing steroids. it obviously suggests that there is a lesser chance of getting a positive biopsy result in patients having a biopsy after 4 weeks of steroid therapy (31). there is considerable debate on the length of specimen needed to confirm gca. gca is a chronic granulomatous inflammation with skip lesions interspersed with normal artery sections. there was a general statement in all biopsy negative results: ‘as gca is a patchy disease, we seemedj 2018, vol 2, no. 2 treatment of giant cell arteritis 43 southeastern european medical journal, 2018; 2(2) cannot exclude arteritis due to the existence of skip lesions in gca.’ skip lesions were seen in 8.5% of specimens with active vasculitis in a previous study (32). the biopsy specimen is divided into 3-4 segments and each visualised under the microscope individually. various studies have shown that the median specimen lengths for positive biopsies were ranging between 5mm and 12 mm but the established benchmark has been 1 cm and above (33). in another study a length of 20 mm or longer had a 2.8 times more likelihood of having gca compared to those less than 20 mm in length (34). in our series, 30 specimens were at least 1cm long prior to fixation. forty specimens were shorter than 1cm or were not adequate samples. there is a degree of contraction of the biopsy specimen before fixation with formalin. it can extend to 12% of contraction for gca positive specimens and 22% for gca negative specimens. the length of the specimen is calculated by measuring the in-vivo with the exvivo size before fixing. if this is true then a specimen of 10mm would contract by more than 2mm if negative for gca (35). complicated cranial gca carries a high risk of visual loss and cerebrovascular accidents. the provision for ophthalmological review and imaging of the head needs to be readily available. in evolving visual changes, the recommendation is to administer intravenous gc for the first 2-3 days in hospital and then change to oral treatment to prevent the catastrophe of visual loss (26, 36). high dose gc treatment must continue until the individual’s inflammatory phase is well controlled. tapering regimes of gc therapy are proposed in the british, european and french consensus guidelines (26, 37, 38). numerous studies on various biological and non-biological agents show prolonged remission and reduction of recurrent relapses. methotrexate has been shown to reduce the dose of gc required in patients with recurrent disease and in those which cannot take high dose gcs (7, 39). recently tocilizumab has been recommended in relapsing gca in the british national institute of clinical excellence guidelines (40). table 2. proposed pathway for the management of temporal arteritis (cont) diagnosis: clinical  age >50  abrupt onset headache (usually unilateral in the temporal area)  scalp pain or difficulty in combing hair  visual symptoms (blurring loss/diplopia)  jaw/tongue claudication  systemic symptoms of fever, weight loss, loss of  appetite, depression and tiredness  symptoms of polymyalgia rheumatica  limb claudication poor prognostic symptoms:  features predictive of ischaemic neuro-ophthalmic complications  jaw claudication  visual symptoms (amaurosis fugax, and diplopia). examination:  abnormal superficial temporal artery: may be tender, thickened with reduced/absent pulsation  scalp tenderness  transient or permanent visual loss (partial or complete) in 20% patients  visual field defect seemedj 2018, vol 2, no. 2 treatment of giant cell arteritis 44 southeastern european medical journal, 2018; 2(2)  relative afferent papillary defect on swinging flashlight test  anterior ischaemic optic neuritis (pale, swollen optic disc with haemorrhages)  central retinal artery occlusion  upper cranial nerve palsies.  features of large vessel gca: asymmetry of pulses and blood pressure and bruits (usually of the upper limb) duplex ultrasound(dul)  clinic review with characteristic ‘halo’ sign laboratory investigations:  markers of a raised inflammatory response  esr and crp, anaemia, thrombocytosis, raised alkaline phosphatase, raised α1 and α2 globulins.  however, temporal arteritis can occur in the face of normal inflammatory markers, if the clinical picture is typical tab  confirmation of clinical diagnosis mri/fdg pet/ct  suspected proximal disease differential diagnosis:  herpes zoster, migraine, cluster headache, acute angle glaucoma, tmj pain, cervical spondylosis, malignancy management of gca:  acute complicated gca (cranial symptoms and signs)  visual ischemia hospital admission with intravenous high dose gc 3 days high dose oral gc  visual loss oral 60 mg prednisolone to protect contralateral eye  uncomplicated gca prednisolone 60mg tapering dose of gc low dose gc +/methotrexate/tocilizumab for recurrent relapse or gc tapering steroid withdrawal:  consider steroid reduction only in the absence of clinical symptoms, signs and lab abnormalities suggestive of active disease.  this should be balanced against the need to use the lowest effective dose, patient wishes and steroid side effects.  suggested tapering regimen: 40-60mgs prednisolone continued until symptoms and laboratory abnormalities resolve (at least 3 to 4 weeks) then dose reduction by 10mg every 2 weeks to 20mg, then by 2.5mg every 2-4 weeks to 10 mg, then by 1mg every 1-2 months provided there is no relapse. seemedj 2018, vol 2, no. 2 treatment of giant cell arteritis 45 southeastern european medical journal, 2018; 2(2) table 3. proposed pathway for the management of temporal arteritis (cont) after care:  rheumatology specialist review  screening for cardio-vascular disease, hypertension and hyperlipidaemia  bone protection and proton pump inhibition if indicated.  systemic symptoms, limb claudication or persistently high-inflammatory markers despite adequate glucocorticosteroid therapy. pet and mri  ta patients screening for abdominal aortic aneurysms (repeat every 2 years whenever indicated). monitoring of therapy:  clinical assessment + inflammatory markers (evidence of relapse, disease-related complications and glucocorticosteroid-related complications)  jaw and tongue claudication.  visual symptoms.  vascular claudication of limbs, bruits and asymmetrical pulses.  polymyalgia symptoms.  osteoporotic risk factors and fractures.  perform the following investigations: at each visit: full blood count, esr/crp, urea and electrolytes, glucose. every 2 years: chest radiograph to monitor for aortic aneurysm (echocardiography, pet and mri may also be appropriate). bone mineral density may be required. management of relapse:  return of symptoms of gca, ischaemic complications, unexplained fever or polymyalgia symptoms.  return of headache  higher dose of glucocorticosteroids.  jaw claudication requires 60 mg prednisolone.  eye symptoms need the use of either 60 mg prednisolone or iv methylprednisolone.  assessment of large vessel involvement. for recurrent relapse:  early introduction of methortrexate or alternative immunosuppressant  in recurrent relapse or failure to wean glucocorticosteroid dose. these immunosuppressive agents should be started at the third relapse.  tocilizumab has been approved for temporal arteritis ophthalmological considerations  aspirin, ophthalmology review for ischemic symptoms/signs  bone protection / ppi  omeprazole, alendronic acid,  calcium and vitamin d conclusion temporal artery biopsy is useful for the diagnosis of cranial gca. the various imaging modalities especially duplex ultrasonography are an important adjunct to the clinical diagnosis. the treatment should not be delayed if there is good clinical suspicion or with risk of visual loss. acknowledgement. none disclosure funding. no specific funding was received for this study. competing interests. none to declare seemedj 2020, vol 4, no. 1 stigmatization of patients with viral infections and mass psychogenic illness 46 southeastern european medical journal, 2020; 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[ocular findings and differential diagnoses in giant cell arteritis (arteriitis cranialis)]. z rheumatol. 2009; 11. dummer w, zillikens d, schulz a, bröcker eb, hamm h. scalp necrosis in temporal (giant cell) arteritis: implications for the dermatologic surgeon. clin exp dermatol. 1996; 12. jeeva i, sajid j, ali o, bonthron dt, frossard pm. atypical takayasu arteritis: a family with five affected siblings. med sci monit. 2007; 13. kötter i, henes jc, wagner ad, loock j, gross wl. does glucocorticosteroid-resistant large-vessel vasculitis (giant cell arteritis and takayasu arteritis) exist and how can remission be achieved? a critical review of the literature. clinical and experimental rheumatology. 2012. 14. salvarani c, crowson cs, o’fallon wm, hunder gg, gabriel se. reappraisal of the epidemiology of giant cell arteritis in olmsted county, minnesota, over a fifty-year period. arthritis care res (hoboken). 2004; 15. gonzalez-gay ma, martinez-dubois c, agudo m, pompei o, blanco r, llorca j. giant cell arteritis: epidemiology, diagnosis, and management. current rheumatology reports. 2010. 16. t. ness, t.a. b, w.a. s, p. l. the diagnosis and treatment of giant cell arteritis. deutsches arzteblatt international. 2013. 17. hunder gg, bloch d a, michel b a, stevens mb, arend wp, calabrese lh, et al. the american college of rheumatology 1990 criteria for the classification of giant cell arteritis. arthritis rheum. 1990; 18. weyand cm, goronzy jj. immune mechanisms in medium and large-vessel vasculitis. nature reviews rheumatology. 2013. 19. loddenkemper t, sharma p, katzan i, plant gt. risk factors for early visual deterioration in temporal arteritis. j neurol neurosurg psychiatry. 2007; 20. m. g. recent advances in giant cell arteritis. curr rheumatol rep. 2018; 21. cid mc, cebrián m, font c, coll-vinent b, hernández-rodríguez j, esparza j, et al. cell adhesion molecules in the development of inflammatory infiltrates in giant cell arteritis: inflammation-induced angiogenesis as the preferential site of leukocyte-endothelial cell interactions. arthritis rheum. 2000; 22. cetinkaya a, kersten rc, brannan pa, thiagarajah c, kulwin dr. intraoperative predictability of temporal artery biopsy results. ophthal plast reconstr surg. 2008; 23. yoon mk, horton jc, mcculley tj. facial nerve injury: a complication of superficial temporal artery biopsy. american journal of ophthalmology. 2011. 24. bhatti mt, goldstein mh. facial nerve injury following superficial temporal artery biopsy. dermatol surg. 2001; 25. rison ra. branch facial nerve trauma after superficial temporal artery biopsy: a case report. j med case rep. 2011; 26. dasgupta b, borg fa, hassan n, alexander l, barraclough k, bourke b, et al. bsr and bhpr guidelines for the management of giant cell arteritis. rheumatology. 2010. seemedj 2020, vol 4, no. 1 stigmatization of patients with viral infections and mass psychogenic illness 47 southeastern european medical journal, 2020; 4(1) 27. miller d v., maleszewski jj. the pathology of large-vessel vasculitides. clinical and experimental rheumatology. 2011. 28. nesher g. the diagnosis and classification of giant cell arteritis. j autoimmun. 2014; 29. roncato c, allix-béguec c, brottiermancini e, gombert b, denis g. diagnostic performance of colour duplex ultrasonography along with temporal artery biopsy in suspicion of giant cell arteritis. clin exp rheumatol. 2017; 30. younge br, cook be, bartley gb, hodge do, hunder gg. initiation of glucocorticoid therapy: before or after temporal artery biopsy? mayo clin proc. 2004; 31. narváez j, bernad b, roig-vilaseca d, garcía-gómez c, gómez-vaquero c, juanola x, et al. influence of previous corticosteroid therapy on temporal artery biopsy yield in giant cell arteritis. semin arthritis rheum. 2007; 32. poller dn, van wyk q, jeffrey mj. the importance of skip lesions in temporal arteritis. j clin pathol. 2000; 33. ypsilantis e, courtney ed, chopra n, karthikesalingam a, eltayab m, katsoulas n, et al. importance of specimen length during temporal artery biopsy. br j surg. 2011; 34. sharma ns, ooi jl, mcgarity bh, vollmer-conna u, mccluskey p. the length of superficial temporal artery biopsies. anz j surg. 2007; 35. su gw, foroozan r, yen mt. quantitative analysis of temporal artery contraction after biopsy for evaluation of giant cell arteritis. can j ophthalmol. 2006; 36. hayreh ss, biousse v. treatment of acute visual loss in giant cell arteritis: should we prescribe high-dose intravenous steroids or just oral steroids? journal of neuro-ophthalmology. 2012. 37. dejaco c, singh yp, perel p, hutchings a, camellino d, mackie s, et al. 2015 recommendations for the management of polymyalgia rheumatica: a european league against rheumatism/american college of rheumatology collaborative initiative. ann rheum dis. 2015; 38. bienvenu b, ly kh, lambert m, agard c, andré m, benhamou y, et al. management of giant cell arteritis: recommendations of the french study group for large vessel vasculitis (gefa). rev med interne. 2016; 39. buttgereit f, dejaco c, matteson el, dasgupta b. polymyalgia rheumatica and giant cell arteritis a systematic review. jamajournal am med assoc. 2016; 40. stone jh, tuckwell k ds et al. efficacy and safety of tocilizumab in patients with giant cell arteritis: primary and secondary outcomes from a phase 3, randomized, double-blind, placebo-controlled trial acr meeting abstracts. arthritis rheum. 2016. seemedj 2018, vol 2, no. 2 attitudes of health care professionals 23 southeastern european medical journal, 2018; 2(2) original article attitudes of health care professionals about importance of healtheducation work of public health nurses in osijek-baranja county 1 marko maceković 1, nada prlić 2 1 medical school osijek 2 faculty of medicine osijek, university of osijek, osijek, croatia corresponding author: marko maceković, markomacekovic@gmail.com received: aug 28, 2017; revised version accepted: mar 10, 2019; published: mar 31, 2019 keywords: public health, nurse, health education, education abstract aim: to examine the attitudes of health care professionals towards the importance of healtheducation work of public health nurses directed at individuals, families and communities. methods: 142 subjects participated in the research, of which 56 public health nurses, 44 primary care (family) physicians and 42 nurses working in family medicine teams. research was conducted at the health centres in beli manastir, osijek, valpovo and đakovo, croatia. a segment of the standardized questionnaire public health nursing survey instrument – a table of interventions conducted by public health nurses (california public health nursing investigation – center for california health workforce studies) – was used as the survey instrument. results: there were no significant differences in the attitudes of respondents by gender (p = 0.898) and age (p = 0.067) regarding the importance of public health nurses’ health-education work. however, respondents aged 60 and over expressed more disagreement with some of the statements related to emotional components of their attitudes (p = 0.019). regarding the length of work experience, there were no statistically significant differences (p = 0.228) on the overall scale of attitudes about health-education work of public health nurses. regarding individual components on the scale, respondents with less work experience tended to agree more with the statements related to emotional components of their attitudes (p = 0.004). regarding the level of education, there were no significant differences in the attitudes of respondents (p = 0.156) towards nurses’ health-education work. research also showed that there were no significant differences in attitudes about the importance of public health nurses’ health-education work when it comes to the subjects’ workplace (p = 0.159). conclusion: health professionals have positive attitudes about health-education work of public health nurses directed at individuals, families and communities. (maceković m, prlić n. attitudes of health care professionals about importance of health-education work of public health nurses in osijek-baranja county. seemedj 2018; 2(2); 23-33) seemedj 2018, vol 2, no. 2 attitudes of health care professionals 24 southeastern european medical journal, 2018; 2(2) introduction health education is an indivisible part of health care, intertwined with human, cultural, social, health, emotional, ethical and psychological needs (1). it is defined as a health care measure for improvement and prevention of health and treatment and mitigation of consequences of diseases, which is achieved by adopting a healthy lifestyle and eliminating harmful health behaviours, as well as by providing education and spreading information on health care (2). national league of nursing education has developed a health education planning model called precede, which clearly defines the role of nurses in the process of health education (2). the model was created because neglecting the essential elements of public health work – preventive and educational tasks – was considered a weakness in the nurse education system. nurses’ health-education work demands cooperation on all levels of health care (primary, secondary, tertiary). besides cooperating at all levels of health care, public health nurses also need to cooperate with other institutions – cities, counties, media, schools, nurseries on matters related to nursing activities. in their work, public health nurses encounter many obstacles and unknowns when it comes to activities related to conducting health-education work. cooperation in croatia is still not established at a satisfactory level. in their research, reckinger et al. evaluated 6 factors of competence: evaluation competencies, individual/family/community competencies, systems competencies, partnership/collaboration competencies, planning competencies and assessment competencies. research was conducted on a sample of 2,269 public health nurses. the authors concluded that the abbreviated instrument could facilitate research on the relation between competencies, public health nursing interventions and public health nursing outcomes (3). in his paper, while demonstrated the importance of public health nurses’ work through health education and its impact on the result of health care (4). perception of public health nurses, in relation to their present role and future activities, was studied by schoenfeld and macdonald. their results showed that public health nurses are focused mainly on working with individuals and families. in addition, they include community care and health promotion as one of the activities (5). also, the study conducted by grumbach, miller, mertz and finocchio contains a survey of public health nurses regarding interventions targeted at individuals, families, communities and the entire system. results indicate that the population health focus of public health nursing is not reflected in the practice activities, management priorities or educational preparation of public health nurses (6). in their study, schaffer, olson, keller and reckinger outline the activities of public health nurses that contribute to community health as one of the recognised factors of their work. some of the important activities addressed in the research were emergency preparedness, health education of individuals and families, receiving and making of referrals, health promotion programs and case management (7). based on a review of relevant literature, thomas et al. conducted a study aimed at determining the effectiveness of interventions conducted by public health nurses. the conclusion was that current methods for a systematic review of quantitative literature can be successfully adapted for issues related to public health nursing and that systematic literature reviews are useful for policy development, program planning and development of future research questions (8). the main purpose of the present study is to examine health care professionals’ attitudes about the importance of public health nurses’ health education work targeted at individuals, families and communities by their level of education, age, gender, work experience and workplace. materials and methods a cross-sectional study involving 142 respondents was conducted. 56 public health nurses, 44 family physicians and 42 nurses working in family medicine teams participated in this survey. the research was conducted at the health centres in beli manastir, osijek, valpovo and đakovo from may to june 2016. the original seemedj 2018, vol 2, no. 2 attitudes of health care professionals 25 southeastern european medical journal, 2018; 2(2) survey questionnaire contained 32 questions. the research instrument used in the survey was a part of the “phn survey instrument” – the table of interventions (california public health nursing investigation – center for california health workforce studies) (6) – which was modified for this research with prior permission for use. the questionnaire was adjusted for the purpose of conducting the research and specifying the work of public health nurses in croatia, with the help from a professor from the department of nursing, medical ethics and palliative medicine of the faculty of medicine osijek. the questionnaire was translated into croatian by an independent language expert. the first part of the survey questionnaire contained demographic issues (age, gender, level of education, length of work experience and place of work). the second part of the survey contained a questionnaire consisting of 19 statements on health education which were rated by public health nurses. the statements were divided into three groups. the first group consisted of five statements related to the cognitive component of an attitude. the second group consisted of six statements related to emotional components of an attitude. the remaining eight statements were related to the statements constituting the behavioural component of attitudes (table 1). table 1. statements statements 1completely disagree 2disagree 3neither agree nor disagree 4agree 5completely agree 1. public health nurses contribute to the quality of health care by providing health education and information in cooperation with other experts at the local community level. 1 2 3 4 5 2. public health nurses contribute to the preservation of health by implementing preventive measures through health education in cooperation with other communitybased institutions. 1 2 3 4 5 3. health education must be integrated into the daily activities of public health nurses. 1 2 3 4 5 4. public health nurses carrying out health education activities succeed in preserving and improving health and preventing illness and injuries at the local community level. 1 2 3 4 5 5. public health nurses establish and monitor health risks in the community, maintain and improve health, prevent diseases and injuries at the system level. 1 2 3 4 5 6. public health nurses feel that they achieve something worthwhile through health education and awareness, cooperation and expert support at the system level (local or state). 1 2 3 4 5 7. public health nurses believe that by cooperating and coordinating health and non-health services they solve or mitigate specific issues by implementing specific knowledge and skills. 1 2 3 4 5 seemedj 2018, vol 2, no. 2 attitudes of health care professionals 26 southeastern european medical journal, 2018; 2(2) 8. public health nurses feel useful when they participate in the implementation of health education measures for the improvement of the quality of health services at the system level. 1 2 3 4 5 9. public health nurses try to implement preventive health measures targeted at individuals/families by providing health education. 1 2 3 4 5 10. public health nurses are confident of the implementation of community-level preventive-health measures, including health education activities. 1 2 3 4 5 11. public health nurses try to provide health education and awareness through the media whenever possible. 1 2 3 4 5 12. public health nurses assess an individual's/family's health by coordinating the individual's health care and education to improve the quality of their life. 1 2 3 4 5 13. public health nurses in their daily work advocate the interests of users/patients by educating them. 1 2 3 4 5 14. public health nurses carry out planned interventions at homes for patients with chronic diseases and gives advice to chronic patients. 1 2 3 4 5 15. public health nurses plan and carry out health consultations and educate families and/or individuals in specific cases. 1 2 3 4 5 16. public health nurses implement health education measures aimed at individuals/families by identifying their problems/needs. 1 2 3 4 5 17. public health nurses detect risk groups timely and implement community-based preventive-health measures. 1 2 3 4 5 18. public health nurses provides information on which rights an individual/family can obtain at the local community level. 1 2 3 4 5 19. public health nurses provide information about the rights and support that individuals/families can obtain at the system level for the purpose of ensuring full health care. 1 2 3 4 5 statements were rated on the likert scale from 1 (completely disagree) to 5 (completely agree). reliability coefficient for the entire scale was calculated. cronbach’s alpha was 0.944. prior to statistical data processing, the respondents were divided into 5 groups based on to their gender, level of education, work position, age and work experience. before the research was conducted, written consents of the ethical committees of the health centres beli manastir, osijek, valpovo and đakovo were obtained. approval to use the standard questionnaire “phn survey instrument” was obtained from kevin grumbach from the ucsf department of family and community medicine of the sf general hospital in san francisco. all respondents were informed about seemedj 2018, vol 2, no. 2 attitudes of health care professionals 27 southeastern european medical journal, 2018; 2(2) the purpose of the research. prior to research, the respondents received a written statement and consent document for participating in the research. all respondents participated in the research voluntarily, which was confirmed by their signature. filling out the questionnaire was anonymous. the research was conducted in accordance with ethical principles and human rights in research. statistical methods data in the categories was represented by absolute and relative frequencies. numerical data was described by median and interquartile range boundaries because the variables did not follow normal distribution. the normality of the distribution of numeric variables was tested by the shapiro-wilk test. differences in numeric variables were tested by the kruskal-wallis test based on the subjects’ level of education and their workplace due to a deviation from the normal distribution. the correlation rating was given by the spearman's coefficient of correlation rho (ρ). all p values were two-tailed. the level of significance was set to α = 0.05. the medcalc statistical program (version 16.2.0, medcalc software bvba, ostend, belgium) was used for the statistical analysis. results the study involved 13 (9.2%) men and 129 (90.8%) women. regarding the level of education (completed secondary education, baccalaureate or university education), the fewest respondents had secondary education qualifications (n=42; 29.6%). regarding work position (public health nurses, nurses in family medicine teams and family medicine physicians), the fewest respondents, 42 (29.6%) of them, were nurses working in family medicine teams. the respondents were divided into the 5 following age groups: up to 29 years of age, 30 – 39 years of age, 40 – 49 years, 50 – 59 years, and 60 years and over. regarding the length of work experience, the respondents were divided into 4 groups: up to 14 years, 15 – 24 years, 25 – 34 years, and 35 years and more. the mean value of the respondents’ age was 48 (interquartile range from 30 to 56, ranging from 24 to 65). as for the length of service, the mean value was 23 years (interquartile range from 5.5 to 33.5 years), ranging from 0.8 to 45 years. all respondents were from the osijek-baranja county. concerning cognitive components on the scale of attitudes in general, the largest number of respondents (n=102; 71.8%) agreed with the statement that health education must be integrated into the daily activities of public health nurses. 14 respondents (9.9%) neither agreed nor disagreed with the statement that public health nurses establish and monitor community health risks, maintain and improve health and prevent diseases and injuries at the system level. there were no significant differences in the rating of cognitive components on the scale of attitudes by gender (mann-whitney u test, p = 0.052) or age group (kruskal-wallis test, p = 0.082). respondents aged between 30 and 39 gave lower ratings for the statement that public health nurses succeed in preserving health by implementing preventive measures through health education in cooperation with other community-based institutions. however, there were no significant differences in comparison with other age groups (kruskal-wallis test, p = 0.060). this is also true for the statement that public health nurses carrying out health education activities succeed in preserving and improving health and preventing illness and injuries at the local community level. however, there were no significant differences between different age groups (kruskal-wallis test, p = 0.216). respondents aged between 30 and 39 and between 50 and 59 gave a lower rating for the statement that public health nurses who determine and monitor health risks in their work maintain and improve health in the community or prevent diseases and injuries at the system level. however, there were no significant differences when compared to other age groups (kruskal-wallis test, p = 0.454) or with regard to work experience (kruskal-wallis test, p = 0.516). the median value of cognitive components of attitudes with regard to the nurses’ work position seemedj 2018, vol 2, no. 2 attitudes of health care professionals 28 southeastern european medical journal, 2018; 2(2) was 4.8 (interquartile range 4.2 to 5). the value was lower for nurses working in a family medicine team (interquartile range = 4.6), but the difference was not significant in comparison with public health nurses (interquartile range = 5) or family medicine practitioners (interquartile range = 4.7). likewise, subjects with a bachelor’s degree gave higher ratings for cognitive components (interquartile range = 5), but the difference was not significant in comparison to the subjects with secondary education qualifications (interquartile range = 4.6) or university qualifications (interquartile range = 4.6). as far as emotional components of attitudes are concerned, 132 (93%) subjects agreed with a statement that public health nurses try to implement preventive health measures directed at individuals/families by carrying out health education activities. among these statements, the most substantial disagreement was expressed for the statement that public health nurses try to conduct health education and educate people through media whenever possible, with which 12 respondents (8.4%) disagreed. significantly lower rating for the statement that public health nurses are confident of implementing preventive health measures on the community level, including health education activities, was given by the subjects above 50 years of age (kruskal-wallis test, p = 0.004). however, there were no significant differences between age groups in relation to other statements. there were also no significant differences in the overall ratings on the scale of emotional components of attitudes by gender (mann-whitney u test, p = 0.723). respondents with 35 or more years of work experience (kruskal-wallis test, p = 0.005), gave significantly lower ratings for said statement, as well as for the statement that public health nurses try to conduct health education activities and educate people through media whenever possible (kruskal-wallis test, p = 0.041). nurses working in family medicine teams gave a significantly lower rating for the statement that public health nurses try to implement preventive health measures directed at individuals/families by providing health education when compared to the ratings given by public health nurses and family medicine practitioners (kruskal-wallis test, p = 0.029). regarding other statements, there were no significant differences considering their work position (kruskal-wallis test, p = 0.395) or level of education (kruskalwallis test, p = 0.492), taking into consideration the overall ratings on the scale of emotional components. with regard to behavioural components, 89 respondents (62.7%) agreed with the statement that public health nurses carry out planned interventions at homes for patients with chronic diseases and give advice to chronic patients. on the other hand, eight respondents (5.7%) disagreed with the statement that public health nurses provide information about the rights and support that individuals/families can obtain at the system level for the purpose of ensuring full health care. there were no significant differences in the rating of behavioural components by gender (mann-whitney u test, p = 0.666) and work experience (kruskal-wallis test, p = 0.399). with regard to age, the statement that public health nurses in their daily work advocate the interests of users/beneficiaries by educating them (kruskal-wallis test, p = 0.024) received the highest rating from the respondents aged 40 to 49. on the other hand, respondents aged 50 and over gave significantly lower ratings for the statement that public health nurses detect risk groups timely and carry out community-based preventive health measures (kruskal-wallis test, p = 0.012). regarding nurses working in family medicine teams, there was a significant disagreement with the statement that public health nurses in their daily work advocate the interests of the users/patients (kruskal-wallis test, p = 0.001) and the statement that public health nurses implement health education measures aimed at individuals/families by identifying their problems/needs (kruskal-wallis test, p = 0.010). when compared to family physicians and public health nurses, nurses working in a family seemedj 2018, vol 2, no. 2 attitudes of health care professionals 29 southeastern european medical journal, 2018; 2(2) medicine team expressed a substantial disagreement with the following statements: public health nurses advocate the interests of users/patients by educating them (kruskalwallis test, p = 0.002); public health nurses carry out planned interventions at homes for patients with chronic diseases and give advice to chronic patients; (kruskal-wallis test, p = 0.032); public health nurses plan and carry out health consultations and educate families and/or individuals in specific cases (kruskal-wallis test, p = 0.031); and public health nurses implement health education measures targeted at individuals/families by identifying their problems/needs (kruskal-wallis test, p = 0.030). there were no significant differences in the overall ratings on the scale of attitudes regarding the importance of health-education work of public health nurses by gender, age, work experience, level of education or work position, as presented in table 2. table 2. overall scale of attitudes about the importance of public health nurses’ health-education work based on gender, age, work experience, level of education and work position overall scale of attitudes about health-education work median (interquartile range) p* gender male 4.5 (3.9 – 4.9) 0.898† female 4.5 (4.1 – 4.9) age groups up to 29 4.8 (4.1 – 5.0) 0.067 30 – 39 4.5 (4.0 – 4.8) 40 – 49 4.9 (4.3 – 5.0) 50 – 59 4.5 (3.9 – 4.8) 60 or over 4.4 (3.9 – 4.7) work experience up to 14 4.7 (4.1 – 5.0) 0.228 15 – 24 4.5 (3.9 – 5.0) 25 – 34 4.5 (3.9 – 4.9) 35 and more 4.4 (4.1 – 4.7) level of education secondary education qualifications 4.3 (3.9 – 4.8) 0.156 post-secondary qualifications 4.6 (4.2 – 4.9) university qualifications 4.6 (4.0 – 5.0) work position nurse in a family medicine team 4.3 (3.8 – 4.8) 0.159 public health nurse 4.6 (4.2 – 4.9) primary care (family) physician 4.5 (3.9 – 5.0) *kruskal-wallis test; †mann-whitney u test older respondents expressed more disagreement with the statements related to emotional (p = 0.019) and behavioural (p = 0.034) components of attitudes, as well as with the scale in general (p = 0.035). respondents with less work experience expressed agreement with emotional (p = 0.004) and behavioural components (p = 0.033) of attitudes to a larger seemedj 2018, vol 2, no. 2 attitudes of health care professionals 30 southeastern european medical journal, 2018; 2(2) degree, as well as with the scale in general (p = 0.020). this correlation is weak, but significant (table 3). table 3: assessment of correlation between the subscales and the scale of attitudes of the respondents towards public health nurses’ health education-work based on age and work experience spearman's correlation coefficient (p value) age work experience cognitive components -0.078 (p = 0.357) -0.081 (p = 0.342) emotional components -0.198 (p = 0.019) -0.243 (p = 0.004) behavioural components -0.178 (p = 0.034) -0.180 (p = 0.033) overall scale rating -0.178 (p = 0.035) -0.196 (p = 0.020) discussion demographic data in the study shows that the age of respondents ranged from 24 to 65 years and that the number of male respondents (13 or 9.2%) was, as expected, considerably lower than the number of female respondents (129 or 90.8). it is therefore difficult to make comparisons on the basis of the respondents’ gender. research has shown that the number of men in all segments of public health care is relatively smaller than the number of women working in this area (9). though contrary to the research, this “trend” has recently been decreasing, even in public health nursing. respondents’ age or length of work experience can be among the most important factors in the creation of their general attitude towards the importance of public health nurses’ healtheducation work. the results of this study did not show significant statistical differences in attitudes, but one can notice that older respondents expressed more disagreement with the statements about health-education work of public health nurses, especially with the statements about emotional components of attitudes. on the other hand, the respondents with less work experience tended to agree more with the statements related to emotional components of attitudes, as well as with the statements on the overall importance of public health nurses’ health-education work. also, the results obtained in this research, considering the entire range of attitudes about the importance of public health nurses’ health-education work, did not exhibit any significant differences in said attitudes with regard to the subjects’ age and length of work experience. we assume these evaluations were based on an objective assessment of their own attitudes, but it is possible to question whether the older respondents observed these statements from the perspective of their own work experience and evaluated them on that basis. an extremely large number of respondents agreed with the statement that health education must be integrated into the daily work of public health nurses. likewise, looking at the overall set of attitudes, several respondents stated that public health nurses try to implement preventive health measures targeted at individuals/families by providing health education. in contrast, nurses working in family medicine teams gave significantly lower ratings for that statement due to differences in their workplaces. seemedj 2018, vol 2, no. 2 attitudes of health care professionals 31 southeastern european medical journal, 2018; 2(2) it should be emphasised that many respondents recognised the importance of integrating health-education work of public health nurses into their day-to-day activities. also, in the majority of research pertaining to interventions, competencies and standards of public health nurses’ practice, health education is regarded as one of the essential and major components of public health nurses’ activities (6, 10). eight respondents disagreed with the statement that public health nurses provide information about the rights and support that individuals/families can obtain at the system level for the purpose of ensuring full health care. also, respondents over the age of 50 and with 35 or more years of work experience gave considerably lower ratings for the statement that public health nurses are confident of the implementation of preventive-health measures targeted at the local community, including health education activities. research by schaffer, olson keller and reckinger and research by the washington states nurses association showed that the public health nurses’ activities at the local community level include coordinating health care for their beneficiaries, instructing patients and providing them with necessary information. also, health care activities of public health nurses are aimed at delivering health care to the entire population for the purpose of improving the quality of life, respecting individuals as equal subjects in the health care system, prioritising primary prevention and cooperating with other professionals and interest groups with a view to promote and protect human health (7, 11, 13). overall ratings on the scale of attitudes towards health care showed that 8.5% of respondents expressed disagreement with the statement related to health education and media literacy. it is important to note that a significantly lower rating was given by the respondents with work experience of 35 years and more. although public health nurses and other health care professionals should and indeed do take part in public health campaigns aimed at health preservation, some respondents did not recognise it. results of this survey showed that the majority of respondents agreed with the statement that public health nurses carrying out health education activities succeed in preserving and improving health and preventing diseases and injuries at the local community level. the majority of authors, such as schoenfeld, macdonald, grumbach, miller, mertz, finocchio, schaffer, olson keller and reckinger, state in their research that public health nurses carry out interventions targeted at individuals and that their health-education work is directed towards the entire community. this implies that it is required to assess the situation and plan health education activities with the aim of improving the health of a population in general (3, 5, 6, 7). results of this research showed that a small number of respondents disagreed with the statement that public health nurses detect risk groups timely and implement communitybased preventive health measures. in their research, schaffer et al. listed several negative impacts on the community health from the perspective of public health nurses, which impacts could arise if no preventive health measures were implemented (7). in their research, schoenfeld and macdonald regarded the implementation of community-based preventive health measures as one of the activities carried out by public health nurses (5). research results also show that nurses working in family medicine teams gave significantly lower ratings for the statements related to emotional components of attitudes about public health nurses’ health-education work in comparison with public health nurses and doctors in family medicine. the same applies to the statement that public health nurses intend to implement preventive health measures targeted at individuals/families by providing health education. in the research conducted by keller et al., the most frequent interventions of public health nurses are listed in five groups. these activities relate to screening, coordination of individual health cases, consulting, cooperation with other seemedj 2018, vol 2, no. 2 attitudes of health care professionals 32 southeastern european medical journal, 2018; 2(2) experts and health education, which can refer to both individuals and the community (12). regarding the behavioural component of attitudes about health education in terms of the respondents’ workplace and level of education, nurses working in family medicine teams expressed more disagreement with the statement “public health nurses in their daily work advocate the interests of users/patients by educating them.” and “…implement health education measures aimed at individuals/families by identifying their problems/needs”. interestingly, regarding the respondents’ age, a considerably higher rating for the first statement, i.e. “public health nurses assess an individual's/family's health by coordinating the individual's health care and education to improve the quality of their life”, was given by the respondents aged 40-49. the question arises as to whether age and experience are related to “sensitivity” in terms of patient health care. in the research they conducted, schoenfeld and macdonald state that the education and consultations for individuals and families provided by public health nurses are largely regarded as one of the most important components of their work. likewise, the reduction, recognition, management and control of chronic diseases are regarded as some of the tasks of public health nurses (5). the results of this study also show that nurses working in family medicine teams expressed more disagreement with the following statements related to the behavioural component of attitudes on the public health nurses’ health-education work: “public health nurses carry out planned interventions at homes for patients with chronic diseases and gives advice to chronic patients” and “public health nurses plan and carry out health consultations and educate families and/or individuals in specific cases”. the problem is that a large number of health professionals still assume that the only intervention performed by a public health nurse is blood pressure and blood sugar control; whereas all other interventions related to specific cases remain within the domain of inhome nursing care. the results also show that nurses working in family medicine teams expressed more disagreement with the statement that public health nurses in their daily work advocate the interests of users/patients by educating them, identifying their problems/needs and implementing health education measures directed at individuals/families. in their research, authors grumbach et al. stated that respondents focused mostly on activities targeted at individuals and families as those are some of the most important components of a public health nurse’s work (6). in their research, which was focused on activities in the community, authors olson keller et al. state that public health nurse’s tasks include the following: focus on the entire population, social representation, ethics, cultural diversity, holistic approach, prevention and health promotion and care for vulnerable groups of population (13). considering the overall ratings on the scale of attitudes, there were no significant differences in terms of the importance of the nurses’ health education work regarding the respondents’ level of education and workplace. however, significant number of nurses with secondary education qualifications gave lower ratings for their attitudes towards the importance of healtheducation work. in the research conducted by zahner and greding, the authors proposed raising awareness of the public, health care workers, social organisations and community members regarding the activities and the function of health-education work of public health nurses in order to keep such work visible and recognised, rather than “hidden” (14). this research revealed that the importance health-education work of public health nurses is largely recognised. however, further research on this issue is important for raising awareness among health professionals regarding the primary and basic purpose of public health services as one of the most important constituents of the health care system. it is necessary to include the primary, secondary and tertiary level, which will increase cooperation seemedj 2018, vol 2, no. 2 attitudes of health care professionals 33 southeastern european medical journal, 2018; 2(2) and coordination of delivering health care to individuals, families and the community, as well as improve the organisation of health care teams involved in such non-institutional care. also, more male respondents should be included in future research so that more genderbased comparisons could be drawn. conclusion public health nurses, nurses working in family medicine teams and family medicine doctors have positive attitudes towards the healtheducation work of public health nurses directed at individuals, families and the community. there are no differences in attitudes on the healtheducation work of public health nurses arising from differences in the respondents’ level of education, age, gender, workplace or length of work experience. acknowledgement. i would like to thank all the institutions at which this survey was conducted, as well as all the subjects who participated in the research, for their cooperation. the paper was presented as a master’s thesis at the faculty of medicine of j. j. strossmayer university in osijek on september 21, 2016 disclosure funding. no specific funding was received for this study. competing interests. none to declare references 1. grčić m, rončević t, sindik j. zdravstveni odgoj i uloga medicinske sestre. hrvatski časopis za javno zdravstvo. 2012;8(32):137-42. 2. sindik j, rončević t. metode zdravstvenog odgoja i promocije zdravlja. unaprjeđenje zdravlja i zdravstveni odgoj. dubrovnik: sveučilište u dubrovniku;2014: 122129. 3. reckinger d, cross s, block de, josten lv, savik k. public health competency instrument: scale reduction and reliability of factors. public health nurs. 2013;6(30):566-74. 4. e. while a. are nurses fit for their public health role? int j nurs stud. 2014;9(51):1191-4. 5. schoenfeld bm, macdonald mb. saskatchewan public health nursing survey perceptions of roles and activities. cjph. 2002;6(93):452-6. 6. grumbach k, miller j, mertz e, finocchio. how much public health in public health nursing practice? public health nurs. 2004;3(21):266-76. 7. schaffer ma, olson keller l, reckinger d. public health nursing activities: visible or invisible. public health nurs. 2015;6(32):711-20. 8. thomas bh, ciliska d, dobbins m, micucci s. a process for systematically reviewing the literature: providing the research evidence for public health nursing interventions. worldv evid-based nu. 2004;1(3):176-84. 9. underwood jm, mowat dl, meagherstewart dm, deber rb, baumann ao, macdonald mb, i sur. building community and public health nursing capacity: a synthesis report of national community health nursing study. cjph. 2009;5(100):1-11. 10. issel ml, ashley m, kirk h, bekemeier b. public health nursing job description: are they aligned with professional standards. j public health man. 2012;3(18):1-8. 11. washington states nurses association. public health and public health nursing. wsna executive committee. 2011. available at: https://www.wsna.org/assets/entryassets/nursingpractice/publications/position-paper-onpublic-health-r2.pdf. date of access 8 august 2016. 12. keller lo, strohschein s, lia-hoagberg, schaffer ma. population-based public health interventions: practice-based and evidencesupported. public health nurs. 2004;4(21):45368. 13. keller lo, strohschein s, lia-hoagberg, schaffer ma. cornerstones of public health nursing; public health nurs. 2011; 28(3):249-260. 14. zahner sj, greding qb. public health nursing practice change and recommendations for improvement. public health nurs. 2005;22(5):422-28 seemedj 2017, vol 1, no. 1 pathophysiological mechanisms of takotsubo cardiomyopathy… 27 southeastern european medical journal, vol 1, 2017. pathophysiological mechanisms of takotsubo cardiomyopathy a systematic review 1 drazen mlinarevic1, hrvoje roguljic1,2, iva juric1, petra zebic mihic1, marul ivandic1, marko stupin1,3 1 department for cardiovascular diseases, osijek university hospital, osijek, croatia 2 department of pharmacology, faculty of medicine, josip juraj strossmayer university of osijek, osijek, croatia 3 department of physiology and immunology, faculty of medicine, josip juraj strossmayer university of osijek, osijek, croatia corresponding author: marko stupin, md marko.stupin@gmail.com introduction received: march 29, 2017; revised version accepted: april 13, 2017; published: april 24. 2017 keywords: takotsubo cardiomyopathy, stress-induced, left ventricular dysfunction, catecholamines takotsubo cardiomyopathy (ttc), also known as stress-induced cardiomyopathy, is a condition with left ventricular (lv) dysfunction, most commonly apical ballooning and less frequently abstract takotsubo cardiomyopathy, also referred to as stress-induced cardiomyopathy, is an acute condition associated with transient left ventricular dysfunction. since it can be induced by increased emotional stress (such as losing a loved one or constant anxiety) it is also called the broken heart syndrome. this type of cardiomyopathy occurs in all age groups and both sexes, but it is most common in postmenopausal women. there are several clinical manifestations such as chest pain, sometimes with heart failure, and often with stsegment changes that may present as acute coronary syndrome. it is characterized by absence of coronary artery obstruction, with transient regional wall motion abnormalities and minimal elevation of cardiac enzyme levels. although wall motion abnormalities are reversible in almost all cases, and long-term prognosis is excellent, this condition is important because in the acute phase it may cause sudden cardiac death. mechanisms and cause of this disease still remain unclear. some possible causes of the disorder include: 1) coronary artery vasospasm, 2) microcirculatory dysfunction, 3) transient obstruction of the left ventricular outflow tract, and 4) excessive release of catecholamine, which seems to have the most important role. the aim of this review is to summarize the most important pathophysiological mechanisms that may be responsible for the development of this type of cardiomyopathy. (mlinarevic d, roguljic h, juric i, zebic mihic p, ivandic m, stupin m. pathophysiological mechanisms of takotsubo cardiomyopathy a systematic review. seemedj 2017;1(1);27-39) seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 28 southeastern european medical journal, vol 1, 2017. midventricular or basal dysfunction. it was first described by sato et al. in the early 1990s, who named it takotsubo because the appearance of the lv is reminiscent of the octopus trap (1). the exact cause of this disease is still unclear. ttc clinically manifests with sudden chest pain and dyspnea preceded by emotional or physical stress, which are similar to those in acute coronary syndrome (acs). besides the onset of chest pain, st-segment elevation and increase in creatine kinase and troponin are very common, and it is necessary to exclude obstructive coronary artery disease (cad) (2). different types of lv wall motion abnormalities have been reported – apical, mid-ventricular or basal hypokinesia, dyskinesia or akinesia. beside the lv, right ventricle may also be affected and it is associated with more severe lv dysfunction (3). any form of dysfunction is reversible with resolution achieved in several days or weeks. overall prognosis is favorable, but acute phase can be accompanied with acute heart failure and cardiogenic shock, rupture of the lv, malignant arrhythmias and in worst cases sudden cardiac death (4). for ttc diagnosis, four diagnostic criteria are suggested: 1) new electrocardiogram (ecg) abnormalities (convex st-segment elevation); 2) transient apical dyskinesia or akinesia detected by echocardiography (echo); 3) non-obstructive cad at angiography: 4) absence of myocarditis, pheochromocytoma, head trauma and intracranial hemorrhage or hypertrophic cardiomyopathy (5). the actual incidence of ttc is unknown, but it is considered that prevalence among patients with acs symptoms is 0.7-2.5%, and it is found most commonly in postmenopausal women (6). since no large studies have confirmed the etiology of stress cardiomyopathy, determination of underlying cause is not possible, but it is almost always preceded by exaggerated emotional, physical or mental stress. sudden death of a loved one, traffic accidents, various types of abuse, business failure, endoscopy, sexual intercourse and other are described as potential triggers of ttc. so far, several possible pathological mechanisms have been proposed, including coronary artery vasospasm, coronary microcirculatory dysfunction, myocarditis, obstruction of the left ventricular outflow tract (lvot), abnormal metabolism of free fatty acids in the cardiac apex and catecholamine overload (7). ttc does not require specific treatment; management is primarily empirical and needs to be individualized for each patient (8). this review will explain recent findings about the pathophysiological mechanisms in this type of cardiomyopathy. clinical presentation, diagnosis and prognosis since ttc patients present with chest pain, dyspnea and syncope, it is difficult to differentiate it from acs based on ecg and laboratory findings. last findings suggest that approximately 1-2 % of patients presenting as acs are ultimately identified as ttc (3, 9). there is no age group or sex that cannot be affected, but there is female predominance (more than 80% of patients are postmenopausal women) and it affects older adults more frequently (1016). similar to acs, ttc patients also have cardiovascular comorbidities such as smoking, hypertension and dyslipidemia (17). ttc is often, but not always, triggered by emotional or physical stress such as receiving bad news, unexpected death of relatives, dissatisfaction with relationships, devastating financial loss or acute medical illness. some of the physical stressors include major surgeries like trauma surgery, infections or neurologic conditions. patients with psychiatric or neurologic disorders may be predisposed to stress cardiomyopathy. virtually any stressor, evena minor one, can be a precipitant of stress cardiomyopathy. it is worth mentioning that no stressor is identified in up to one-third of patients. physical findings are nonspecific and often normal, but the patient may present with signs of acs or acute congestive heart failure. other symptoms include nausea, vomiting and palpitations. cardiac bradyarrhythmias and tachyarrhythmias, including ventricular tachycardia and ventricular fibrillation, may seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 29 southeastern european medical journal, vol 1, 2017. develop. (10, 11, 18-20). some patients may develop signs of heart failure, and approximately 10% of patients may develop cardiogenic shock (21-23). left ventricular outflow tract obstruction caused by left ventricular basal hyperkinesis produces latepeaking systolic murmur and can contribute to severe mitral regurgitation, hypotension and shock (24). like any patient in whom acs is suspected, ecg should be the initial test obtained. ecg abnormalities are common in patients with ttc (25). most common abnormalities on initial ecg are st-segment elevation and t-wave inversion. studies found that st-segment elevations involve the precordial leads and are maximal in leads v2-v3 (26). patients with ttc have a significantly lower amplitude of st segment elevation compared to stemi from lad occlusion. st depression is a less common finding in patients with ttc. kosuge et al. found that combination of absent abnormal q waves, absent reciprocal changes, lack of st-segment elevation in lead v1, and the presence of stsegment elevation in lead avr had 91% sensitivity and 96% specificity for ttc. other possible findings include qt interval prolongation and non-specific ecg abnormalities. however, all these criteria have imperfect diagnostic accuracy and are not reliable for differentiation between the two conditions in the emergency setting to guide their management (e.g. decision to undergo emergency coronary angiography) (27). serum cardiac troponin i levels (tni) are elevated in 90% of patients with ttc, while creatine kinase levels are generally normal or slightly elevated. the brain natriuretic peptide (bnp) orn-terminal pro-bnp are elevated in most patients with ttc (25). in patients with ttc, mean tni level at the time of admission has been reported as moderately elevated. nascimento et al. used this finding to create a criterion for differentiating between stemi and ttc (28). the troponin ejection fraction product (tefp) is the product of the peak troponin i level and the echocardiographically acquired ejection fraction. a tefp ≥250 had an overall accuracy of 91% for stemi identification. budnik et al. found that the ntprobnp / tni ratio was capable of distinguishing between ttc and stemi. in this study, the concentration of ntprobnp was greater in patients with ttc than in ones with stemi, while the concentration of tni and ckmb mass was higher in the stemi group than in the ttc group (29). several studies analyzed the levels of circulating catecholamines in the acute phase and found that 75% of ttc patients had higher levels than patients with stemi, but their role in diagnosing ttc is unclear (30,31). wall motion abnormalities are best identified by echocardiography or left ventriculography. trans-thoracic echocardiography (tte) is used as a quick method of diagnosing wall-motion abnormalities typical for tcc (32), such as hypokinesis or akinesis of the mid-segment and apical segment of the lv, which is present in 81.7% of patients (25). crucially, these wallmotion abnormalities extend beyond the distribution of any single coronary artery. lv ejection fraction was found to be 20-49% on admission. the resolution of ttc usually occurs within four weeks with lvef improving to 5976%. ttc diagnosis is usually confirmed by coronary angiography. acute presentation with stsegment elevation and symptoms suggestive of acs mandate immediate evaluation with coronary angiography to exclude coronary occlusion. the prognosis in tcm is excellent in most cases, with nearly 95% of patients experiencing complete recovery within 4-8 weeks (13). mortality estimates range from 1% to 5.9%. complications occur in around 20% of patients and include lv outflow obstruction, heart failure, ventricular arrhythmias, mitral regurgitation, lv mural thrombus formation or death. international takotsubo registry reported 5.9% mortality after 30 days (33). the rate of mortality during longterm follow-up was 5.6% per patient-year. a swedish registry study found a 30-day mortality of 4.1% in 302 patients with ttc (34). these trials compared mortality in ttc with matched cohorts of patients with acute myocardial infarction or acute coronary syndrome and found a similar risk of death. seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 30 southeastern european medical journal, vol 1, 2017. coronary vasospasm and microcirculatory dysfunction coronary vasospasm was the first pathophysiological process considered as a cause ttc in the original article by sato et al. in 1991 (35). since ttc usually presents with transient wall motion abnormalities (“stunned myocardium”) covering the irrigation territories of several coronary arteries (36), a multivessel coronary vasospasm could be the potential cause. however, according to current literature, coronary vasospasm is not a likely cause, because spontaneous vasospasm is rare in these patients (37) and cannot be induced in all the patients during angiography (38). finally, ttc has a specific histological phenotype which differs from stunning associated with coronary artery disease (39). on the other hand, there is an increasing body of evidence that microvascular dysfunction is one of potential pathophysiological mechanisms of disease. diffuse microcirculatory dysfunction could explain wall motion abnormalities (wmas) in several myocardial regions. microcirculatory dysfunction may be primary or secondary, i.e. caused by excess of circulating cateholamines (37). the consequence of microcirculatory dysfunction is coronary slow flow (csf), which in turn causes myocardial wmas (40). several methods have been used for evaluating csf – doppler guidewire during angiography (41), timi (thrombolysis in myocardial infarction) myocardial perfusion grade (tmpg) (42, 43) and timi frame count (tfc) (11,44). the most commonly used method is tfc which is defined as “the number of frames required for the contrast material to travel from coronary ostium to the standardized distal landmark” (44). several studies have demonstrated an increased tfc (i.e. slower coronary flow) in ttc patients (45-47), which supports the role of microcirculatory dysfunction. a study by martin et al. in 2013, using peripheral arterial tonometry, demonstrated increased vascular reactivity and decreased endothelial function in response to acute mental stress in patients with previous ttc (48). although most of the available literature supports the role of either primary or secondary microcirculatory dysfunction in ttc, it is important to acknowledge that the results are not always uniform. a recent retrospective study by khalid et al. found the tfc to be higher in left anterior descending (lad) coronary artery of ttc patients, but no difference was found in tfc in right (rca) or circumflex (cx) coronary artery (45). this anatomical distribution could explain the most common form of ttc involving the apex and the midventricular subtype, but not the less frequent form which involves the basal myocardium. sharkey et al., in 2008, found a modest increase in tfc in ttc patients compared to controls (acute anterior stemi with lad occlusion), which was statistically significant in lad and cx, but not in the rca (30). abe et al. (2003) found no coronary slow flow, no abnormalities using doppler guidewire technique and no evidence of viral myocarditis in a series of 17 patients (49). collste et al. (2015) investigated coronary flow reserve (cfr) by dobutamine stress echocardiography and the authors could not induce microcirculatory dysfunction, but found cfr at low-dose dobutamine was significantly lower in patients with tsc compared to controls (50). metabolic disturbance one possible hypothesis is that ttc may be considered a metabolic form of cardiomyopathy with disturbed cardiomyocyte metabolism. several studies that were researching metabolic changes in stunned myocardium found alterations in glucose and fatty acid uptake. those alterations may be the result of primary metabolic disturbance in cardiomyocyte or due to mitochondrial disturbance (51, 52). the metabolic disturbance was likely linked to the sudden preceding stress and resulted in corresponding perfusion abnormalities. yoshida et al. describe abnormalities in coronary perfusion and severe myocardial metabolic disorder in patients with ttc based on the results of thallium-201 myocardial single-photon emission computed tomography (spect) and f18 fluorodeoxyglucose (f-18 fdg) myocardial seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 31 southeastern european medical journal, vol 1, 2017. positron emission tomography (pet). they noticed markedly decreased uptake of f-18 fdgon pet at the apical region while thallium 201 images showed only mildly reduced uptake. reason for decreased uptake may be due to increased density of beta receptors noted in apex (53).several studies have reported prolonged reduction and reduced uptake of f18 fdg in patients subjected to multiple cycles of ischemia and reperfusion (54, 55). still, the precise mechanism for reduced glucose uptake in stunned myocardium remains unknown. lvot obstruction & myocarditis according to earlier studies, lvot obstruction is registered in 15-25% of patients with ttc (10, 56) and kawaji et al detected it in 33% of patients (57). although it was proposed as a possible pathophysiological mechanism of the disease, it remains uncertain whether it is a consequence rather than a cause of stress cardiomyopathy (37, 58). some researchers claim that there is much evidence indicating this relation actually exists. transient dynamic lvot gradient was detected at initial evaluation in a substantial proportion of patients described by tsuchihashi et al. (1) and other investigators (24, 59). at least in some patients, a possible mechanism for ttc could be a dynamic lvot obstruction preceding the ischemic event. some of those patients, primarily women, may have geometric predisposition to dynamic lvot obstruction, such as sigmoid or bulging interventricular septum (60, 61), reduced left ventricular volume (62-64) or abnormal mitro-aortic and septoaortic angles (65), which may manifest only in the setting of intense adrenergic stimulation or hypovolemia (37). elderly women have a higher tendency to develop hypertrophy of the basal anterior septum. the angle of the septum may cause increase in the speed in the outflow tract which simulate a hypertrophic cardiomyopathy (66). it is also associated with an abnormal orientation of the mitral valve due to flaccidity, deformity of valve, false chordae, disturbances of the papillary muscles, or systolic anterior movement (67-69) with mitral regurgitation. it is known that even in a normal heart, exposure to an exogenous catecholamine, such as dobutamine infusion, can precipitate dynamic lvot obstruction (70). if present, the dynamic obstruction increases apical lv wall stress and lv filling pressure, increasing myocardial oxygen demand at the mid-to-apical cavity. if this persists, apical hypoperfusion and ischemia may occur, with regional wall motion abnormality and stunning. increased adrenergic tone might produce primary lvot obstruction leading to secondary ischemia and focal wall-motion abnormalities. physical or emotional stress could be the trigger of acute development of lvot obstruction, which could produce severe apical ischemia. identification of acute dynamic lvot obstruction as the possible initial mechanism in some of the patients with stress cardiomyopathy may have important clinical and therapeutic implications (71). previously suggested possible role of a transient dynamic lvot obstruction in the pathogenesis of this syndrome is not strongly supported by other investigators. according to ishihara, it is unlikely that lvot obstruction is the cause of ttc because most of these patients do not have lvot obstruction. it is known that this condition is characterized not only by reduced apical lv wall motion, but also hyperkinesis of the basal lv wall, and that the combination possibly causes the lvot obstruction (57, 72, 73). lvot obstruction is not a prerequisite, but can contribute in a deteriorating clinical course of ttc (71, 73). takotsubo cardiomyopathy is characterized not only by reduced apical lv wall motion, but also hyperkinesis of the basal lv wall. this combination causes the lvot obstruction in ttc. the suggested possibility that myocarditis leads to transient lv dysfunction and takotsubo cardiomyopathy is not well supported by the data. arguments to rule out myocarditis include absence of typical clinical signs, unspecific findings on myocardial biopsy and negative results on serum tests for viral serology. some studies used cardiac magnetic resonance seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 32 southeastern european medical journal, vol 1, 2017. imaging, which has shown no evidence of myocarditis (35, 74-77). catecholamines – pathophysiological hallmark of stress cardiomyopathy exposure to high intensity stress conditions, whether physical or emotional, has been associated with most cases of ttc (37). furthermore, patients with pheochromocytoma, a catecholamine-producing tumor, are prone to develop a similar form of cardiomyopathy (78). in the animal model of subarachnoid hemorrhage, a condition with heightened catecholamine levels, a correlation between the extent of myocardial damage and sympathetic discharge was reported (79). those observations suggest increased sympathetic activity and catecholamine mediated effect as a crucial factor for development of the broken heart syndrome. although apart from catecholaminemediated effect other theories about pathophysiology of stress cardiomyopathy coexist, the current mayo clinic criteria require catecholamine-producing tumor to be ruled out for establishing a ttc diagnosis (80). relationship between high levels of serum catecholamines and stress in patients suffering from cardiomyopathy was first shown in 2003 (76). abraham et al. reported the emergence of all morphologic forms of ttc in patients exposed to catecholamines and other betareceptor agonists used routinely during procedures and diagnostic tests (81). study of wittstein et al. reported that levels of catecholamines and dopamine are approximately two to three times higher in patients with ttc in comparison with cardiomyopathy caused by acute myocardial infarction (77). interestingly, the correlation between morphological changes of the left ventricle in ttc and distribution of adrenoreceptors has been observed. a majority of β2 receptors with negative inotropic effect is in the apex of the left ventricle where ballooning process takes place which is consistent with a theory of catecholaminergic stress (39). moreover, increased release of the catecholamines from the hearts of the patients affected with ttc has been reported (82). some of the nuclear imaging studies also stressed the influence of the sympathetic nervous system in development of ttc. in eight patients with ttc, a decreased 123i-metaiodobenzylguanidine uptake within left ventricle was registered, indicating the existence of cardiac sympathetic hyperactivity as pathophysiological pathway (83). moreover, a concordance between regional wall motion abnormalities and reduced uptake of the glucose and free fatty acid has been shown (84). although this impairment of metabolism is not fully understood, catecholamine induced injury of cardiomyocytes is probably the cause of a metabolic stunned myocardium. experiments in animals provided further evidence regarding the role of the catecholamines in disease pathogenesis. iatrogenic administration of β2 agonists or immobilization stress in animals can result in reversible left ventricular apical ballooning. this adverse effect could be mitigated by αand β-receptor blocking agents (85, 86). stress cardiomyopathy is characterized by similar molecular manifestations as the other catecholamine-mediated cardiomyopathies. those morphological alterations caused by catecholamine overload include: extracellular matrix overproduction, contraction band necrosis and mononuclear cell infiltration (37). catecholamine induced accumulation of collagen α-1 (i) chain in extracellular matrix results in large and rapid increase in fibrosis. high levels of catecholamine may result in high levels of profibrotic mediators (angiotensin ii and free oxygen radicals), which can activate stimulating connective tissue growth factor, transforming growth factor β and the profibrotic osteopontin (87). catecholamine overload stimulates β-adrenoreceptors and alters the expression of calcium-regulatory protein genes which cause alteration of the calcium regulatory system (88). sarcolipin (sln) and phospholamban (pln) are proteins of sarcoplasmic reticulum (sr) which regulate cardiac contractility. sln regulates the sarcoplasmic/endoplasmic reticulum calcium atpase 2 (serca2) by lowering its affinity for seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 33 southeastern european medical journal, vol 1, 2017. calcium. in acute phase of ttc, ventricular expression of sln is raised and could contribute to contractile dysfunction (89). at physiological conditions, binding norepinephrine on β1 – adrenoreceptors (β1ar) and epinephrine on β2 adrenoreceptors (β2ar) in cardiomyocytes results in positive inotropic response. in normal human ventricular myocardium, there are four times more β1ar than β2ar (90). positive inotropic response is the result of β1ar or β2ar activating stimulatory g protein (gs) family, which activates protein kinase a (pka) pathway reflected as an increased contractile response (91). supraphysiological levels of catecholamines result in β2-coupling from gs to inhibitory g protein (gi), which is reflected as a negative inotropic effect. this process is also called stimulus trafficking (92). the density of β2ar is the highest in cardial apex, so there is the greatest negative inotropic effect (91). the β2ar -gi protein pathway can activate the p38 mitogen-activated protein kinase (mapk) alteration of myofilament sensitivity. β2ar–gi protein has a favorable outcome on stress cardiomioypathy by stimulating the pi3k–aktsignaling pathway, which activates antiapoptotic genes (nfκb1 and bcl2) (37). this is a physiological balance because β1ar–gs protein pathway has the proapoptotic effect (93). it is cardioprotective because it minimalizes catecholaminergic stimulation. after epinephrine levels are normalized, β2ar–gi switch to β2ar–gs or are degraded, which results in recovery of cardiomyocyte contractile function (91). figure 1. schematic overview of pathophysiological mechanisms involved in catecholamine-mediated takotsubo cardiomyopathy. seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 34 southeastern european medical journal, vol 1, 2017. although pathophysiological pathway of ttc is still unclear, it is certain that catecholamine overload presents a common denominator in development of the broken heart syndrome, as presented in figure 1. despite plentiful clinical findings, further research is obligatory to complete the puzzle of this rare but potentially severe disease. conclusion takotsubo cardiomyopathy is an important type of acute heart failure with transient left ventricular wall motion abnormalities. the symptomatology, echocardiographic and electrocardiographic features frequently mimic acute coronary syndrome, which is why ttc must be considered in a differential diagnosis of patients with acute chest pain. according to contemporary 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(1999) opposing effects of beta(1)and beta(2)-adrenergic receptors on cardiac myocyte apoptosis: role of a pertussis toxin-sensitive g protein. circulation 100: 2210–2212 seemedj 2019, vol 3, no. 2 nutrition habits influence parkinson’s disease clinical presentation 11 southeastern european medical journal, 2019; 3(2) original article do nutrition habits influence the clinical presentation of parkinson’s disease?1 svetlana tomic 1, 2, vlasta pekic 1, 2, 3, zeljka popijac 1, tomislav pucic 1, marta petek vinkovic 1, 2, zvonimir popovic 1, 2, bojan resan 2, 4, tihana gilman kuric 1, 2 1 clinical department of neurology, osijek clinical hospital centre, osijek, croatia 2 faculty of medicine, josip juraj strossmayer university of osijek, osijek, croatia 3 faculty of dental medicine and health, josip juraj strossmayer university of osijek, osijek, croatia 4 school of engineering, university of applied sciences northwestern switzerland, windisch, switzerland corresponding author: svetlana tomic, svetlana.tomic@vip.hr received: jun 15, 2019; revised version accepted: dec 6, 2019; published: dec 16, 2019 keywords: parkinson`s disease, nutrition, dairy products, protein, fruit, vegetables abstract introduction: parkinson's disease (pd) is the second most common neurodegenerative disorder characterized with alpha-synuclein pathology. for the majority of patients, except for some genetic forms, etiology is still unknown. there are some implications that food intake and gut microbiota could contribute to pd. aim: the aim of this paper is to analyze the influence of protein, fruit and vegetable intake on the clinical presentation of idiopathic parkinson disease patients and methods: patients with idiopathic pd were surveyed for demographic data and nutritional habits in regards to protein, fruit and vegetable intake. motor symptoms were evaluated using the unified parkinson disease rating scale (updrs) part iii and iv, cognitive impairment using mini mental state examination (mmse) and depression using beck depression inventory (bdi). results: we have analyzed data of 96 patients. patients using fewer dairy products have more often tremor type of pd (p<0.040). we did not find any differences in severity of motor symptoms, disease stage, age when disease start, frequency of motor complications and fluctuation of therapy, depression and cognitive impairment according to protein, fruit and vegetable ingestion. conclusion: higher intake of dairy products could influence the appearance of less favorable forms of parkinson's disease (rigor type). protein, fruit and vegetable intake do not influence the disease appearance, severity of motor symptoms, motor fluctuation and complication of therapy, disease stage, the appearance of cognitive impairment nor depression in parkinson's disease patients. (tomic s, pekic v, popijac z, pucic t, petek vinkovic m, popovic z, resan b, gilman kuric t. do nutrition habits influence the clinical presentation of parkinson’s disease?. seemedj 2019; 3(2); 1121) seemedj 2019, vol 3, no. 2 nutrition habits influence parkinson’s disease clinical presentation 12 southeastern european medical journal, 2019; 3(2) introduction parkinson's disease (pd) is the second most common neurodegenerative disorder characterised by alpha-synuclein pathology [1]. for the majority of patients, the etiology of the disease is still unknown. some recent studies in pd patients are addressing food and its influence on gut microbiota [2]. it is known that pd patients have altered gut microbiota with abundance in proinflammatory and reduction in anti-inflammatory microbes [3]. those changes can promote alpha-synuclein pathology in the enteric nervous system, which could spread in a prion-like manner to the brain [4]. many studies have been done in order to explore the influence of various types of nutrition on the risk of parkinson's disease. there have been papers reporting an increased risk associated with diets rich in animal fat [5,6], dairy foods [7,8], raw meat [9], and carbohydrates [10], while some other studies have not found a strong correlation between nutrition and the risk of pd [2, 11-13]. it has been shown that some nutrients could decrease the risk of developing pd in many ways. monounsaturated (mufas) and polyunsaturated fatty acids (pufas) are known to have anti-inflammatory effects and they can reduce oxidative stress and inhibit apoptosis [14,15]. vitamin a, b6, b9, b12, d and e have been proven to have protective effects and decrease the risk of pd [16-20]. early post-treatment (after 6hydroxydopamine toxicity) with retinoic acid in the animal model is able to provide protection from neurodegeneration in nigrostriatal dopaminergic neurons [16]. decreased levels of vitamins b6, b9 and b12 lead to elevated levels of homocysteine. hyperhomocysteinemia damages the dna, depletes energy reserves and subsequently induces neuron apoptosis [21]. calcitriol (vitamin d) reduces neuronal toxicity, while vitamin e has the ability to reduce mptpinduced (1-methyl-4-phenyl-1,2,3,6tetrahydropyridine) toxicity in dopaminergic neurons [22,23]. using food with anthocyanin and proanthocyanidin-rich substances improves the mitochondrial function and reduces the level of neurodegeneration [24]. this paper aims to analyse the influence of protein, fruit and vegetable intake on the clinical presentation of idiopathic parkinson’s disease observed through several parameters: the age when the disease was diagnosed, severity of motor symptoms, type of parkinson`s disease, appearance of motor fluctuations and complication of therapy, depression and cognitive impairment. patients and methods the study was conducted on patients with idiopathic parkinson’s disease diagnosed during a regular check-up at the outpatient clinic at osijek clinical hospital centre. their consent of participation in this study was obtained and the study was approved by the local ethics committee. the patients were surveyed for age, sex, disease duration, the age when the disease was diagnosed and nutrition habits pertaining to protein, fruit and vegetable intake for 3 months prior to the examination (part of the mini nutritional assessment, source nestlé nutrition institute). motor symptoms were evaluated using the unified parkinson disease rating scale (updrs) part iii (range from 0 to 103 points) and iv (range from 0 to 23 points), cognitive impairment symptoms were evaluated using the mini mental state examination (mmse; range from 0 to 30 points) and depression was evaluated using the beck depression inventory (bdi; range from 0 to 63 points). according to the updrs part iii, patients were divided into 2 groups – rigor-dominant and tremor-dominant. categorical data were presented as absolute frequencies and percentages, while the differences between nominal variables were tested by the fisher's exact test. numerical data were tested with the shapiro-wilk test for normality of data distribution. afterwards, numerical data were presented with the mean and standard deviation in the case of normal, and with the median and interquartile range in the case of abnormal distribution. the comparison between nominal and numerical variables with normal distribution was tested using the one-way anova or student’s t-test, seemedj 2019, vol 3, no. 2 nutrition habits influence parkinson’s disease clinical presentation 13 southeastern european medical journal, 2019; 3(2) while for numerical variables with abnormal distribution, the mann-whitney and kruskalwallis tests were used. statistical significance was defined as α=0.05, while the statistical analysis was conducted with statistica 13 (statsoft inc., tulsa, oklahoma, usa). results a total of 96 patients were analysed, of whom 57 (59.4%) males and 39 (49.6%) females, with the mean age of 70.22±8.598. regarding the type of parkinson’s disease, 50 (52.1%) patients had the tremor-dominant type, while 46 (47.9%) patients had the rigor-dominant type. table 1 shows the data about disease duration, disease stage, the age of onset, updrs iii, mmse and bdi score. table 1. demographic data, updrs iii, mmse, bds data mean/median std. dev./25th75th min/max age 70.22 ±8.598 48/80 disease duration 4.00 (2.00-10.00) 1/16 age when disease started 64.64 ±9.730 36/81 updrs iii 16.50 (10.25-23.75) 3/83 mmse 26.00 (23.00-28.00) 15/30 bds 13.60 ±8.992 0/44 updrs iii – unified parkinson`s disease rating scale part iii; mmse – mini mental state examination; bds – beck table 2 presents the frequency of nutrition intake. the majority of the patients eat meat every day (40.6%) or every other day (37.5%). more than two-thirds (71.9%) of the patients consume dairy products every day, while 68.8% of them consume two or more legumes and eggs per week. half of them eat meat, fish and poultry every day and more than 2 pieces of fruit and vegetable per day (table 2). seemedj 2019, vol 3, no. 2 nutrition habits influence parkinson’s disease clinical presentation 14 southeastern european medical journal, 2019; 3(2) table 2. frequency of protein, fruit and vegetable intake food n % protein intake  once per week  twice per week  every other day  everyday 4 17 36 39 4.2 17.7 37.5 40.6 dairy products one per day  yes  no 69 27 71.9 28.1 legumes or eggs two or more per week  yes  no 66 30 68.8 31.2 meet, fish, poultry every day  yes  no 46 50 47.9 52.1 fruit or vegetable ≥2 per day  no  yes 43 53 44.8 55.2 the updrs iii and iv results were compared with the nutrition habits of pd patients, but no significant difference was found. the comparison is shown in table 3. table 3. difference in updrs part iii and motor complications and fluctations of therapy according to protein, fruit and vegetable intake updrs iii „off“ dyskinesia protein intake  once per week  twice per week  every other day  everyday median p yes/no (n) p yes/no (n) p 19.50 22.00 17.00 15.00 0.580 1/3 10/7 15/21 17/22 0.572* 0/4 2/15 5/31 9/30 0.614* dairy products one per day  yes  no 17.00 16.00 0.925 30/39 13/14 0.820* 11/58 5/22 0.766* legumes or eggs two or more per week  yes  no 17.00 14.50 0.351 29/37 14/16 0.828* 11/55 5/25 1.000* meet, fish, poultry every day  yes  no 15.00 18.50 0.334 20/26 23/27 0.840* 10/36 6/44 0.275* fruit or vegetable ≥2 per day  no  yes 17.00 16.00 0.487 19/25 24/28 1.00* 6/37 10/42 0.656* differences in the age of disease diagnosis, mmse and bdi score regarding the patients’ nutrition habits were also observed, but there were no statistically significant differences, as shown in table 4. seemedj 2019, vol 3, no. 2 nutrition habits influence parkinson’s disease clinical presentation 15 southeastern european medical journal, 2019; 3(2) table 4. difference in age when disease start, type of parkinson`s disease (tremor vrs rigor), mmse and bdi according to protein, fruit and vegetable intake age when disease started type of pd mmse bdi protein intake  once per week  twice per week  every other day  everyday mean p t/r (n) p median p mean p 68.00 68.71 64.86 62.31 0.126 2/2 8/9 22/14 18/21 0.574* 25.50 24.00 26.00 26.00 0.179 18.50 16.00 12.53 13.05 0.274 dairy products one per day  yes  no 65.29 62.96 0.295 31/38 19/8 0.040* 26.00 25.00 0.993 14.45 11.44 0.072 legumes or eggs two or more per week  yes  no 65.27 63.23 0.344 35/31 15/15 0.828* 25.00 27.00 0.348 12.82 15.33 0.388 meet, fish, poultry every day  yes  no 62.24 65.00 0.704 22/24 28/22 0.540* 26.00 25.00 0.323 13.28 13.90 0.991 fruit or vegetable ≥2 per day  no  yes 63.60 65.31 0.399 23/21 27/25 0.835* 26.00 25.00 0.219 14.09 12.56 0.383 pd – parkinson`s disease; t/r – tremor vrs. rigor type; mmse mini mental state examination; bdi – beck depression inventory the analysis of influence of nutrition habits of pd patients showed that the tremor-dominant group of pd patients consumed dairy products less frequently, while there were no statistically significant differences in other nutrition habits of patients with tremor-dominant and rigordominant parkinson's disease (table 5). table 5. comparison between type of parkinson disease and nutritional habits of parkinson disease patients type of pd seemedj 2019, vol 3, no. 2 nutrition habits influence parkinson’s disease clinical presentation 16 southeastern european medical journal, 2019; 3(2) t/r (n) p protein intake  once per week  twice per week  every other day  everyday 2/2 8/9 22/14 18/21 0.574* dairy products one per day  yes  no 31/38 19/8 0.040* legumes or eggs two or more per week  yes  no 35/31 15/15 0.828* meet, fish, poultry every day  yes  no 22/24 28/22 0.540* fruit or vegetable ≥2 per day  yes  no 23/21 27/25 0.835* discussion nutrition habits of 96 pd patients were surveyed for the purpose of analysing their influence on the clinical presentation of the disease. the majority of the patients ingested protein in the form of dairy products, eggs and legumes and half of them in the form of meat, fish and poultry on a daily basis. no difference in the age when the disease was diagnosed, severity of motor symptoms, disease stage, frequency of motor fluctuations and complication of therapy, nor depression and cognitive impairment associated with protein, fruit and vegetable intake was found. it was only found that the patients consuming fewer dairy products more frequently suffer from tremor-dominant pd, which is a form of the disease with a favourable outcome [25]. neuropathological studies have shown that the rigor-dominant type compared to the tremor type of pd exhibits a higher level of neuronal loss of the locus coeruleus, lateral and medial part of the substantia nigra with more severe gliosis, extra-neuronal melanin deposits and neuroaxonal dystrophy in the substantia nigra [26]. many papers have reported a relation between dairy products and pd [2730]. there are several possible explanations of how dairy products influence neurodegeneration and the risk of pd. one of the possible explanations is the influence of milk fat on gut microbiota. milk fat (mf) and pufa-rich fat had similar effects on bacteroidetes and firmicutes, but besides this, mf has the ability to greatly increase bilophila wadsworthia. an increased level of bilophila wadsworthia was associated with the pro-inflammatory t helper type 1 (t(h)1) immune response [31]. dairy products also have the ability to reduce the uric acid level, which could cause a greater pd incidence and faster pd progression [32,33]. it is also known that they can induce insulin resistance and that this has an impact on the development of not only pd, but also of alzheimer's disease [34,35]. people suffering from lactose intolerance and consuming dairy products are at the risk of intestinal inflammation and increased intestinal permeability. besides that, milk could be contaminated with neurotoxic pesticides [36]. finally, the ingestion of bovine microbiota could affect human microbiota through small intestinal bacterial overgrowth (sibo), which could increase the risk of pd [37-39]. marczewska et al. found in 45 advanced-stage pd patients that patients with high protein intake experience moderate to seemedj 2019, vol 3, no. 2 nutrition habits influence parkinson’s disease clinical presentation 17 southeastern european medical journal, 2019; 3(2) severe disease motor symptoms more frequently [40]. serum carotenoid, retinol and tocopherol concentrations were lower in pd patients with severe motor symptoms and a more advanced stage of the disease [41]. many studies have proved that high protein intake affects the motor response to levodopa therapy, causing the appearance of motor fluctuations [42], and that the use of a protein-redistribution diet helps with the amelioration of „on-off“ fluctuations [43]. on the other hand, a proteinrestricted diet (prd) has proven to worsen the fluctuations with the worsening of dyskinesia [44]. this negative influence of proteins on the motor function is not present in early stages of pd and usually appears after 13 years of disease duration, or 8 years after levodopa was introduced in therapy [45]. no influence of protein ingestion on motor symptoms, motor fluctuations and complication of therapy was found, probably due to the fact that the study group of patients was mostly in the early stage of the disease (the median was 4.00 years), during which there is no such negative influence. the ingestion of saturated fatty acids, lower consumption of milk and dairy products and consumption of full-fat dairy products have a negative impact on age-related cognitive decline, mild cognitive impairment and vascular dementia [46]. there is limited data about the protective role of fruit and vegetable ingestion on cognitive decline, dementia and vascular dementia [46]. no differences in nutrition habits and cognitive impairment were found. the majority of our patients were diagnosed with mild cognitive impairment (mmse median was 24.00) and were a rather homogeneous group (mmse interquartile range from 23.00 to 28.00). besides this, mmse is not very sensitive to the subcortical type of cognitive impairment that is present in parkinson`s disease [47]. therefore, a more heterogenous patient group with more sensitive tests for subcortical cognitive impairment should be used for more conclusive results. the mind diet (the mediterranean-dash intervention for neurodegenerative delay diet) that emphasises intake of fresh fruit, vegetables, and legumes was not associated with the reduction of depression risk, in contrast to the mediterranean diet [48]. consumption of full-fat yogurt was related to a lower risk of depression, but only in women, in the study of pernezcornago et al [49]. there is evidence (in largescale and well-conducted observational studies) that the ingestion of seafood, vegetables, fruit and nuts reduces the risk of depression [50]. unfortunately, no significant differences between nutrition habits and depression were found in this study. higher intake of dairy products could influence the appearance of less favourable forms of parkinson's disease (rigor-dominant type). this study did not find any influence of protein, fruit and vegetable intake on the age of disease diagnosis, severity of motor symptoms, disease stage, motor fluctuations and complications of therapy, appearance of cognitive impairment or depression in parkinson's disease patients. the limitation of this study is a small sample size. furthermore, only the source of protein intake (meat, legumes or eggs, dairy) was analysed, while there are no evaluated sources regarding fruit and vegetables. for that reason, the data about the ingestion of fruit and vegetables producing antioxidant effects are limited. therefore, the interpretation of our study results must be taken with caution. ethical approval: “all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards.” informed consent: “informed consent was obtained from all individual participants included in the study. additional informed consent was obtained from all individual participants for whom identifying information is included in this article.” acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. seemedj 2019, vol 3, no. 2 nutrition habits influence parkinson’s disease clinical presentation 18 southeastern european medical journal, 2019; 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revised version accepted: nov 6, 2022; published: nov 28, 2022 keywords: postexercise cutaneous blood flow, endurance exercise, short-lasting aerobic exercise, postexercise hypotension abstract aim. there are numerous reports of attenuation of cutaneous blood flow (cbf) during the recovery period after a single bout of exercise, but no one has investigated the cbf response to consecutive short-lasting aerobic exercise (slae) sessions following exhaustive endurance exercise (eee) on daily basis, although this is a commonly used training regime in recreational athletes and could cause a cumulative increase in cbf that may be important for wound healing. this study examined the effects of eee on forearm skin blood flow (ldf), cutaneous vascular conductance (cvc), and mean arterial pressure (map) in response to slae sessions performed for 7 days after eee, as well as the correlation between cutaneous blood flow and indices associated with heart rate (hr) to examine the role of thermoregulation in post-exercise hr regulation. methods. in 19 recreational runners, ldf, map, hr, heart rate recovery (hrr), and hr variability indices (lnrmssd, lnhf, and lnlf/ hf) were measured after slae in the form of submaximal graded cycling performed on consecutive days after eee in the form of a half marathon and compared with baseline values before eee. a significant time effect was observed for all measured parameters throughout the study period. results. postexercise ldf increased 24 hours after eee compared with baseline (77.814 au compared with 54.712 au). postexercise hypotension was significantly more pronounced immediately after eee compared with baseline (88.95.3 mmhg compared with 99.33.2 mmhg). however, postexercise cvc showed a progressive increase compared with baseline both immediately and 24 hours after eee (0.53 0.07 au/mmhg, 0.66 0.09 au/mmhg compared with 0.4 0.09 au/mmhg). a small negative correlation between ldf and hrr was observed throughout the experimental protocol. conclusion. these results suggest that eee strongly influences cutaneous and systemic hemodynamics and cardiac autonomic response during recovery after slae. our most important finding was that eee improved cutaneous perfusion 24 hours after completion of eee, which may be important for wound healing. the results of our study are potentially applicable for patients with chronic wounds who should be encouraged to exercise moderately on daily basis and to include endurance exercises occasionally, as this strategy potentiates postexercise cutaneous perfusion. (potočnik n. the effect of submaximal exercise on cutaneous blood flow, thermoregulation and recovery hemodynamics following endurance exercise. seemedj 2022; 6(2); 32-43) seemedj 2022, vol 6, no 2 effect of submaximal exercise 33 southeastern european medical journal, 2022; 6(2) introduction recreational athletes often include exercises of varying intensity and duration in their daily training schedule. during several consecutive training sessions and subsequent recovery, a complex interplay of many physiological processes, such as thermoregulation, autonomic nervous system adaptation, and nitric oxide production (no), establishes a dynamic state that may be reflected in cutaneous blood flow (cbf) as the integration site of all the above processes. the term “training recovery” was introduced by bishop et al. as recovery between successive exercise sessions (1). it can be assumed that the cbf response in training recovery is the cumulative result of recovery and exercise, especially when exhaustive endurance exercise (eee), with its long-term effects on cardiovascular function (2), is included in the training process. there are numerous reports of cbf in the recovery phase after a single exercise session (3–7) but no one has investigated the cbf response to successive submaximal, short-lasting aerobic exercise (slae) sessions following exhaustive exercise on daily basis, although this is a commonly used training regime in recreational athletes. knowledge of the cbf response to cumulative exercise could be useful in planning exercise strategies for patients with chronic wounds. during exercise, cutaneous blood flow increases in hairy skin (5), as it is mainly controlled by sympathetic vasodilator fibers. in addition, vasodilation is provoked in an attempt to achieve thermal equilibrium and thus stable core body temperature to allow heat dissipation (8). however, during the recovery phase after exercise, with a sudden decrease in metabolic demand, loss of muscle pump activity, and significant change in autonomic nervous system activity (ans), cutaneous blood flow rapidly decreases to near baseline levels (5). nevertheless, core (9,10) and muscle (9) temperatures remain elevated 60-90 min after exercise cessation. cutaneous blood vessels in non-glabrous skin are innervated by two branches of sympathetic nerves: noradrenergic vasoconstrictor and cholinergic vasodilator fibers. consequently, the rapid decrease in cutaneous blood flow after cessation of exercise could be caused by increased vasoconstriction, active vasodilation, or a combination of both. because nitric oxide mediates 30-45% of active cutaneous vasodilation (11), it could potentially contribute to the modulation of cutaneous blood flow after exercise. after exercise, heart rate (hr) decreases to preexercise levels. the initial, rapid phase of hr recovery caused by parasympathetic reactivation is followed by a secondary, slow decline mediated by continued parasympathetic reactivation and sympathetic withdrawal (2). several studies have examined the mechanisms underlying these post-exercise autonomic responses and have suggested that exercise-induced thermoregulatory demands play an important role (12). there is evidence for the role of thermoregulation in post-exercise hr recovery (13). a possible link between thermoregulation and hr recovery after exercise could be related to thermoregulatoryinduced changes in systemic (mean arterial pressure (map)) and/or skin hemodynamics (4). recently, it has been shown that hr recovery after a short slae in response to eee differs significantly (2), synchronous with changes in ans activity. thus, it can be assumed that cutaneous blood flow is simultaneously affected. therefore, the aim of this study was to investigate the effects of eee on cutaneous blood flow in hairy skin after subsequent slaes and the relationship between the changes in hr and cutaneous hemodynamic responses to exercise. therefore, heart rate, arterial blood pressure, and cutaneous blood flow in the volar forearm were measured simultaneously during recovery from slae, repeated sequentially before and immediately, 24 hours, 48 hours, and 7 days after eee. we hypothesized that eee provokes an increase in cutaneous perfusion and that skin blood flow correlates with concomitant heart rate changes after exercise. seemedj 2022, vol 6, no 2 effect of submaximal exercise 34 southeastern european medical journal, 2022; 6(2) methods subjects nineteen recreational runners (13 men) aged 40.4 ± 15.2 years with a body mass index of 23.0 ± 2.7 were recruited to participate voluntarily in this study, which was in accordance with the declaration of helsinki and approved by the national ethics committee of the republic of slovenia (on 10/ 19/2021/no. 0120126/2021/10). written informed consent was obtained from all subjects participating in the study. physical examination and medical history revealed no autonomic dysfunction, chronic diseases, medication use, or smoking. their ecg and arterial blood pressure were normal. individual maximum heart rate (hrmax) was determined according to the formula (14). procedure the study was conducted in an air-conditioned laboratory room between 2 pm and 6 pm. subjects refrained from physical exertion at least three days before the first exercise test and were asked not to perform any other physical activity during the experimental period. they were not allowed to consume alcohol, caffeine, or tobacco for at least 2 hours before the start of each exercise test and were asked to eat a light meal 1 hour before visiting the laboratory. each participant visited the laboratory 4 times a week for 5 measurements. on the first visit, eee was performed in the form of a 21-km run on the predefined outdoor track, on partly sunny days with temperatures between 17°c and 20°c and humidity between 55% and 65%. experimental sessions. slae was repeated at 5 different time points: before, one hour (day 0), 24 hours (day 1), 48 hours (day 2), and 7 days (day 7) after eee, as shown in figure 1a. values recorded in the "before" session are referred to as baseline values. slae consisted of a 5-minute rest in a seated position followed by a graded exercise test on the ergoselect 100 (ergoline, germany) cycloergometer, starting at 40 w for 3 minutes and continuing with a load increase of 50 w every 3 minutes until the target heart rate (hrpeak), defined as 85% of the individual hrmax, was reached (fig. 1b). immediately thereafter, subjects terminated exercise and remained in a seated position for an additional 15 min to passively recover from slae. figure 1. experimental protocol: schedule of successive exercise bouts (a); short-lasting graded cycling protocol (b) measurements ecg and arterial blood pressure on the middle finger of the right hand were measured continuously (finometer, amsterdam, netherlands). during the measurement, the right arm and hand were placed in an arm rest when sitting on the cycle ergometer that was adjusted to the height of the heart and equipped with hemodynamic sensors. cutaneous blood flow was measured by laser doppler (ldf) perfusion monitoring (periflux system 5000, perimed, stockholm, sweden) on the right volar forearm. the principles of ldf monitoring are described elsewhere (15). all signals were recorded simultaneously at 500 hz using windaq data acquisition software (dataqinstruments inc., ohio, zda). at each slae, a time interval of 180 s between the 12th and 15th minutes after cessation of cycling was selected for further analysis. averaged ldf and map were determined using nevrokard software seemedj 2022, vol 6, no 2 effect of submaximal exercise 35 southeastern european medical journal, 2022; 6(2) (nevrokard, slovenia). cvc was calculated as the ratio of ldf to map. heart rate analysis. rr interval duration was determined beat by beat from ecg using ahrv_file_preparation software (nevrokard, slovenia), with artefacts and premature beats corrected. heart rate (hr) and root mean square of successive differences (rmssd) as timedomain markers of parasympathetic modulation (task force of the european society of cardiology) were calculated from rr interval time series using ahrv_analysis_software (nevrokard), and the natural logarithm of rmssd (lnrmssd) was calculated. the following frequency-domain parameters were determined using the autoregressive method (nevrokard, slovenia): the power of the highfrequency band (hf; 0.15-0.40 hz), a marker of parasympathetic modulation, and the lf/hf ratio, which is considered a marker of sympathovagal balance, where lf denotes the power of the low-frequency band (0.04-0.15 hz) (16). hf and lf/hf were expressed in the natural logarithmic scale (lnhf) heart rate recovery in 30 (hrr30) and 60 s (hrr60) was defined as the differences between hrpeak and hr, measured 30 and 60 s after termination of slae for each slae session. statistical analysis statistical analysis was performed using ibm spss statistics, version 27 (ibm, new york, usa). data were tested for normality and log transformed if they were not normally distributed. we compared the mean differences between the measured parameters over time (before, day 0, day 1, day 2, and day 7) with a oneway repeated-measures analysis anova (ranova). the assumption of sphericity was checked with the mauchly's test; greenhousegeisser or huynh-feldt corrections were applied when the sphericity assumption was violated, as published elsewhere (17). when a significant time effect was detected, appropriate contrast tests were used to detect differences between mean values at day 0, day 1, day 2, and day 7 compared with the corresponding baseline (before) values. for post hoc comparisons, the least significant difference test was applied and bonferroni correction was used to eliminate type i errors in multiple comparisons. pearson's product-moment correlation coefficients (r) were used to estimate bivariate correlations between each index of cardiac autonomic activity (hrr30, hrr60, lnrmssd, lnhf, and lnlf/ hf) and subsequent ldf. sse performance indices (pmax, hrstmax, rpe) were combined over the entire experimental period. the following criteria were used to interpret the magnitude of correlations: r ≤ 0.1 trivial; r > 0.1-0.3 small; r > 0.3-0.5 moderate; r > 0.5-0.7 large; r > 0.7-0.9 very large (17). data are presented as mean ± standard deviation, and a confidence level p < 0.05 was chosen. results ldf, cvc and map in recovery from subsequent slae, there was a significant time effect found in ldf (f=2.9; p=0.026; η2=0.08), cvc (f=4.5; p=0.003; η2=0.11) and map (f=2.8; p=0.030; η2=0.10), as displayed on figures 2b, 2c and 2a, respectively. ldf was significantly augmented 24 hours after eee compared to baseline (fig. 2b), while cvc was enhanced already one hour after eee cessation and even more 24 hours after eee compared to baseline conditions (fig. 2c). map was significantly decreased one hour after eee cessation (fig. 2a) regarding to baseline. seemedj 2022, vol 6, no 2 effect of submaximal exercise 36 southeastern european medical journal, 2022; 6(2) figure 2: changes in mean arterial pressure (map) (a), laser doppler flow in the forearm (ldf) (b) and cutaneous vascular conductance (cvc) (c) during experimental protocol (before – before exhausting endurance exercise (eee); day 0 – 1 hour after eee; day 1 – 24 hours after eee; day 2 – 48 hours after eee, day 7 – 1 week after eee). * statistically significant compared to baseline (before) p – p value according to ranova hr and hr-derived measures there was a significant time effect found in hr after slae (f=45.6; p<0.001; η2=0.38), hrr30 (f=16.2; p<0.001; η2=0.20) and hrr60 (f=19.6; p<0.01; η2=0.16) across all measuring points as displayed in figure 3. all three parameters exhibited a biphasic change compared to baseline: hr was significantly higher at day 0 and significantly lower at day 1 compared to baseline (fig. 3), yet hrr30 and hrr60 were significantly lower at day 0 and significantly higher at day 1 compared to baseline (fig. 3). additionally, hr after slae was decreased also at day 2 compared to baseline (fig. 3). seemedj 2022, vol 6, no 2 effect of submaximal exercise 37 southeastern european medical journal, 2022; 6(2) figure 3: changes in heart rate (hr) after short-lasting aerobic exercise, heart rate recovery in 30 s (hrr30) and heart rate recovery in 60 s (hrr60) during experimental protocol (before – before exhausting endurance exercise (eee); day 0 – 1 hour after eee; day 1 – 24 hours after eee; day 2 – 48 hours after eee, day 7 – 1 week after eee). * statistically significant compared to baseline (before) p – p value according to ranova the parameters of heart rate variability, lnrmssd (f=10.1; p<0.001; η2=0.19), lnhf (f=7.3; p<0.001; η2=0.13) and lnlf/hf (f=7.6; p=0.011; η 2=0.22) exhibited significant time effect along measuring protocol (fig. 4). lnrmssd behaved biphasically compared to baseline, while lnhf and lnlf/hf differed significantly from baseline only at the day 0 (fig. 4). figure 4: changes in natural logarithm of root mean square of successive differences (lnrmssd), the power of high-frequency band (lnhf) and the relationship between the power of low-frequency band to high frequency band (lnlf/hf) during the measuring protocol. * statistically significant compared to baseline (before) p – p value according to ranova bivariate correlations when all data were pooled there were no clear correlations neither between ldf and hr seemedj 2022, vol 6, no 2 effect of submaximal exercise 38 southeastern european medical journal, 2022; 6(2) (p=0.93), lnrmssd (p=0.66), lnhf (p=0.17) and lnlf/hf (p=0.34) nor between cvc and hr (p=0.17), lnrmssd (p=0.78), lnhf (p=0.42), lnlf/hf (p=0.65), hrr30 (p=0.09) and hrr60 (p=0.18), respectively. a small negative correlation was observed between ldf and hrr30 (p=0.036, r= -0.229) as well as between ldf and hrr60 (p=0.032, r= -0.233), as seen in figures 5a and 5b. figure 5: linear fit between laser-doppler flow (ldf) in the forearm during recovery from shortlasting aerobic exercise and heart rate recovery in 30 s (hrr30) (a) and heart rate recovery in 60 s (hrr60) (b); 95% confidence limits are indicated by a dashed line. p – p value according to ranova; r – pearson’s coefficient; n – number of correlated points discussion in the present study, we investigated the response of cutaneous blood flow in hairy skin in the recovery phase after short lasting aerobic exercise at different time points after exhaustive endurance exercise and compared it with concomitant changes in cardiac autonomic activity in recreational runners. the aim of this study was to contribute to the understanding of the control of cutaneous blood flow in relation to training history. we found that training history has a strong influence on cutaneous (ldf and cvc) and systemic hemodynamics (map), as well as on cardiac autonomic (hr, hr derived indices) response during recovery from slae. our most important finding was that exhaustive endurance exercise as part of the training history improved cutaneous perfusion 24 hours after completion of eee, which may be important for wound healing. the results of the present study are as follows: ldf in the recovery phase at slae after exhaustive endurance training was significantly increased 24 hours after eee compared with baseline, which might be important for wound healing, a progressive increase in cvc after training was observed during the first 24 hours after exhaustive endurance training, post-exercise hypotension was significantly increased immediately after exhaustive endurance training, and a small bivariate correlation between ldf and hrr30(60) was observed throughout the experimental protocol, indicating the role of thermoregulation in post-exercise regulation hr to our knowledge, this is the first study to report time-dependent changes in ldf during exercise recovery after exhaustive endurance training. accordingly, we can only speculate about the mechanisms controlling these changes. during the recovery phase after exercise, there is a rapid decrease in cutaneous perfusion despite a a b seemedj 2022, vol 6, no 2 effect of submaximal exercise 39 southeastern european medical journal, 2022; 6(2) significant residual heat load (18–21). it has been suggested that both centrally mediated (sympathetic adrenergic and cholinergic innervation) and peripheral endotheliumdependent and independent factors may modulate the control of cutaneous blood flow after exercise (9,22) and that either enhanced vasoconstriction or attenuated active vasodilation may play the role (18). since nitric oxide mediates 30-45% of active cutaneous vasodilation (18), it seems likely that the bioavailability of no may additionally influence post-exercise vasodilation. however, the timedependent contributions of all potentially involved mechanisms during recovery after exhaustive endurance training remain unclear. according to the literature, postexercise hypotension is caused by at least two mechanisms: increased vasodilation due to thermoregulatory challenges and/or impairment of baroreceptor reflex (23). the predominant mechanism was found to depend on exercise intensity and duration (24). liu et al. reported that prolonged endurance exercise induces significant postexercise hypotension regardless of exercise intensity (24), and it has been shown that, at least in males, reduced cardiac baroreflex sensitivity triggers postexercise hypotension (25) after a half marathon. in addition, mcginn et al. (26) found decreased baroreflex sensitivity as a major cause of postexercise hypotension after 90 minutes of graded dynamic exercise. surprisingly, ldf, cvc, and map did not change synchronously during the measurement protocol, although this was generally expected. based on cvc, which was increased immediately and 24 hours after eee compared with baseline, one would expect increased postexercise hypotension and skin vasodilation at the same measuring points. however, map was significantly decreased only immediately after eee and ldf was significantly decreased only 24 hours after eee, suggesting that different mechanisms are involved in the regulation of map and ldf. the main cause of the postexercise increase in cvc immediately after eee appears to be a decreased map, whereas its further increase 24 hours after eee is due to an increased ldf at that measurement time point. response immediately after exhaustive endurance exercise immediately after eee, the postexercise decrease of map observed in our study compared with baseline (fig. 2) can be attributed to an impairment of the baroreflex, as suggested by mourot et al. after endurance exercise, core body temperature and mean muscle temperature remain elevated for up to 24 hours (20). therefore, increased cutaneous blood flow during recovery from short lasting aerobic exercise performed immediately after eee, compared with baseline before endurance exercise, would be expected to eliminate the additional heat load generated during eee. however, in our study, postexercise ldf was not increased after eee compared with baseline. the concomitant observed dominance of the cardiac sympathetic nervous system activity over the parasympathetic activity (fig. 4) suggests that the simultaneously increased adrenergic vasoconstrictor nerve activity affecting the skin may prevent vasodilation. another possible explanation would be increased oxidative stress immediately after endurance exercise. numerous data show that a decreased ability to release no in the skin microcirculation is associated with increased oxidative stress (19,27). interestingly, a biphasic response of exhaled no, a potent surrogate for oxidative stress (28), was reported after endurance exercise (29). more specifically, exhaled no increased significantly immediately after endurance exercise and decreased 24 hours after endurance exercise compared with the level before endurance exercise. consequently, we can probably assume that increased oxidative stress immediately after eee induces endothelial dysfunction with the impairment of no availability. immediately after a half marathon, the response of the cardiovascular system to short lasting aerobic exercise has been shown to mimic the response found in cardiovascular dysfunction (2). thus, seemedj 2022, vol 6, no 2 effect of submaximal exercise 40 southeastern european medical journal, 2022; 6(2) subsequent endothelial dysfunction could be due to cardiovascular dysfunction at that time point. dehydration after endurance exercise could lead to decreased map and affect cutaneous blood flow. however, concurrent measurements of total body water showed that water volume was preserved after endurance exercise in our study, as reported elsewhere (2). response 24 hours after exhaustive endurance exercise postexercise map was decreased 24 hours after cessation of endurance training compared with baseline, although not to a statistically significant extent (fig. 2). these results are consistent with the findings of other authors who report that post-exercise hypotension can persist for up to 24 hours (23,30,31). the main finding of our study was that both ldf and cvc increased significantly at slae 24 hours after endurance training compared to baseline (fig. 2). since increased skin blood flow accelerates the wound healing process (32), these results support the use of endurance training in wound healing. this finding of our study can be applied in practice by encouraging patients with chronic wounds to include occasional endurance exercise in their regular moderate training schedule to increase skin blood flow and accelerate wound healing. we can only suggest a few possible mechanisms responsible for the improvement in cutaneous blood flow 24 hours after eee: increased sympathetic cholinergic vasodilation as the main mechanism to eliminate excessive heat, increased no bioavailability due to suppressed oxidative stress 24 hours after eee (29), the involvement of adenosine receptors that have been shown to prevent the decrease in skin blood flow after exercise (18), or a complex interplay of many physiological processes leading to cardiovascular supercompensation, which probably includes improved skin blood flow observed by many authors after endurance training (2,33,34). further studies need to be conducted to substantiate these assumptions. no significant changes in measured parameters were observed on day 2 and day 7, suggesting that the effect of eee on cutaneous blood flow diminishes after two days. bivariate correlations we found a small negative correlation between postexercise ldf and hrr30 and hrr60 when data were pooled across the entire measurement protocol (fig. 5). a correlation of this magnitude requires careful interpretation. however, this correlation suggests that improved cutaneous blood flow delays recovery of hr after slaes regardless of endurance exercise. these results are consistent with the conclusions of pecanha et al. (4), who found that heat stress delays heart rate recovery and highlighted the role of thermoregulation in postexercise regulation of the hr. they suggested that thermally induced redirection of blood flow to cutaneous vessels, as evidenced by increased ldf, may involve reduced central blood volume and ventricular filling during recovery, resulting in compensatory sympathetic activation and parasympathetic deactivation after exercise (4). limitations of our study and perspectives our cohort consists of recreational runners, so results are limited to physically active, healthy individuals. further studies are needed to prove that endurance exercise accelerates wound healing in patients prone to developing chronic wounds, such as those with type 2 diabetes. gender and age differences of cutaneous blood flow and postexercise hypotension after endurance exercise described by stapelton (20) and mourot et al. (25) were not examined in our study. the analysis of our data should be repeated separately for men and women to identify possible gender differences that could influence our conclusions. measurement of core temperature during the study protocol would be helpful to determine the magnitude of heat stress induced by endurance exercise compared with shortlasting exercise. seemedj 2022, vol 6, no 2 effect of submaximal exercise 41 southeastern european medical journal, 2022; 6(2) the proposed mechanisms underlying our results could be better determined if different substances such as bretylium tosylate, an inhibitor of noradrenergic vasoconstriction, and a no synthase inhibitor (lname) were applied to the skin during the measurement protocol. furthermore, spectral analysis based on the fast fourier or wavelet transform of the periodic oscillations of the ldf after exercise could be used to analyze the periodic oscillations of different frequencies representing the influence of heartbeat, respiration, intrinsic myogenic activity, and neurogenic factors, respectively, on cutaneous blood flow after exercise (3,7). additional analysis of ldf should be performed according to spectral analysis guidelines. conclusion we measured the response of cutaneous blood flow in the forearm during recovery after short lasting aerobic exercise at different time points after exhaustive endurance exercise and compared it with concomitant changes in cardiac autonomic activity in recreational runners. we aimed to contribute to the understanding of the control of cutaneous blood flow in relation to training history. we found that training history has a strong influence on cutaneous and systemic hemodynamics as well as on cardiac autonomic response during recovery to slae. our most important finding was that exhaustive endurance exercise as a part of the training history improved cutaneous perfusion 24 hours after completion of eee, which may be important for wound healing. the results of our study are potentially applicable for patients with chronic wounds, who should be encouraged to exercise moderately on daily basis and to occasionally include endurance exercises, as this strategy potentiates postexercise cutaneous perfusion. acknowledgement. this research work was funded by the slovenian research agency (core research funding no. p3-0019). the author declares no conflict of interests. disclosure funding. this research work was funded by the slovenian research agency (core research funding no. p3-0019). competing interests. none to declare. references 1. bishop pa, jones e, woods ak. recovery from training: a brief review. j strength conditioning research. 2008; 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103(2):443-51. doi: 10.1152/japplphysiol.00117.2007 https://pubmed.ncbi.nlm.nih.gov/?sort=date&term=winter+s&cauthor_id=19270035 seemedj 2022, vol 6, no 2 effect of submaximal exercise 43 southeastern european medical journal, 2022; 6(2) 23. farinatti p, pescatello ls, crisafulli a, taiar r, fernandez ab. editorial: postexercise hypotension: clinical applications and potential mechanisms. front physiol. 2022 apr 12;13:720. 24. liu s, thomas sg, sasson z, banks l, busato m, goodman jm. blood pressure reduction following prolonged exercise in young and middle-aged endurance athletes. eur j prev cardiol 2013; 20(6):956-62. doi: 10.1177/2047487312454759. 25. mourot l, fornasiero a, rakobowchuk m, isacco l, brighenti a, stella f, zignoli a, pellegrini b, tarperi c, schena f. postexercise hypotension and reduced cardiac baroreflex after half-marathon run: in men, but not in women. int j environ res public health 2020; 17(17):6337. doi: 10.3390/ijerph17176337. 26. mcginn r, carter mr, barrera-ramirez j, sigal rj, flouris ad, kenny gp. does type 1 diabetes alter post-exercise thermoregulatory and cardiovascular function in young adults? scand j med sci sports 2015; 25(5):e504-14. doi: 10.1111/sms.12344370 27. holowatz la, thompson-torgerson c, kenney wl. aging and the control of human skin blood flow. front biosci (landmark ed 2010; 15(2):718-39. doi: 10.2741/3642. 28. pavord id, beasley r, agusti a, anderson gp, bel e, brusselle g, cullinan p, custovic a, ducharme fm, fahy jv, frey u, gibson p, heaney lg, holt pg, humbert m, lloyd cm, marks g, martinez fd, sly pd, von mutius e, wenzel s, zar hj, bush a. after asthma: redefining airways diseases. lancet. 2018; 391(10118):350–400. 29. sierra ap, oliveira-junior mc, almeida fm, benetti m, oliveira r, felix sn, santos genaro i, mangueira saraiva romanholo b, ghorayeb n, peduti dal molin kiss ma, cury-boaventura mf, pesquero jb, vieira rp. impairment on cardiopulmonary function after marathon: role of exhaled nitric oxide. oxid med cell longev 2019; 2019:5134360. doi: 10.1155/2019/5134360. 30. pescatello ls, buchner dm, jakicic jm, powell ke, kraus we, bloodgood b, et al. physical activity to prevent and treat hypertension: a systematic review. med sci sports exerc 2019; 51(6):1314-1323. doi: 10.1249/mss.0000000000001943. 31. day c, wu y, pescatello ls. evaluating the methodological quality of postexercise hypotension aerobic exercise interventions. front physiol. 2022; 13:407. 32. raposio e, bertozzi n, moretti r, grignaffini e, grieco mp. laser doppler flowmetry and transcutaneous oximetry in chronic skin ulcers: a comparative evaluation. wounds a compend clin res pract 2017; 29(7):190-195. 33. buchheit m, laursen pb, al haddad h, ahmaidi s. exercise-induced plasma volume expansion and post-exercise parasympathetic reactivation. eur j appl physiol 2009; 105(3):471-81. doi: 10.1007/s00421-008-0925-1. 34. hautala a, tulppo mp, mäkikallio th, laukkanen r, nissilä s, huikuri h v. changes in cardiac autonomic regulation after prolonged maximal exercise. clin physiol. 2001; 21(2):238–45.. author contribution. single author https://pubmed.ncbi.nlm.nih.gov/?sort=date&term=cullinan+p&cauthor_id=28911920 https://pubmed.ncbi.nlm.nih.gov/?sort=date&term=custovic+a&cauthor_id=28911920 https://pubmed.ncbi.nlm.nih.gov/?sort=date&term=ducharme+fm&cauthor_id=28911920 https://pubmed.ncbi.nlm.nih.gov/?sort=date&term=fahy+jv&cauthor_id=28911920 https://pubmed.ncbi.nlm.nih.gov/?sort=date&term=frey+u&cauthor_id=28911920 https://pubmed.ncbi.nlm.nih.gov/?sort=date&term=gibson+p&cauthor_id=28911920 https://pubmed.ncbi.nlm.nih.gov/?sort=date&term=heaney+lg&cauthor_id=28911920 https://pubmed.ncbi.nlm.nih.gov/?sort=date&term=holt+pg&cauthor_id=28911920 https://pubmed.ncbi.nlm.nih.gov/?sort=date&term=humbert+m&cauthor_id=28911920 https://pubmed.ncbi.nlm.nih.gov/?sort=date&term=lloyd+cm&cauthor_id=28911920 https://pubmed.ncbi.nlm.nih.gov/?sort=date&term=marks+g&cauthor_id=28911920 https://pubmed.ncbi.nlm.nih.gov/?sort=date&term=martinez+fd&cauthor_id=28911920 https://pubmed.ncbi.nlm.nih.gov/?sort=date&term=sly+pd&cauthor_id=28911920 https://pubmed.ncbi.nlm.nih.gov/?sort=date&term=von+mutius+e&cauthor_id=28911920 https://pubmed.ncbi.nlm.nih.gov/?sort=date&term=von+mutius+e&cauthor_id=28911920 https://pubmed.ncbi.nlm.nih.gov/?sort=date&term=wenzel+s&cauthor_id=28911920 https://pubmed.ncbi.nlm.nih.gov/?sort=date&term=zar+hj&cauthor_id=28911920 https://pubmed.ncbi.nlm.nih.gov/?sort=date&term=bush+a&cauthor_id=28911920 seemedj 2020, vol 4, no. 1 survival of multiple myeloma patients undergoing dialysis 25 southeastern european medical journal, 2020; 4(1) original article survival of multiple myeloma patients undergoing dialysis or plasma exchange a single centre’s 25-year experience 1 petra smajić* 1, ema schönberger 1, vlatka periša 1,2, jasminka sinčić petričević 1,2, lada zibar 1,3, kristina kralik 1 1 faculty of medicine, josip juraj strossmayer university of osijek, croatia 2 university hospital centre osijek, croatia 3 merkur university hospital, zagreb, croatia *corresponding author: petra smajić, smajicpetra@gmail.com received: feb 19, 2020; revised version accepted: mar 18, 2020; published: apr 27, 2020 keywords: multiple myeloma, kidney failure, haemodialysis, plasma exchange, survival abstract aim: multiple myeloma (mm) patients might require haemodialysis (hd) and/or plasma exchange (pe) in cases of acute kidney injury (aki) and/or chronic kidney disease (ckd) and/or other indications. the study analysed the survival of mm patients who required hd and/or pe. subjects and methods: all 144 patients treated for mm at the university hospital centre osijek between 1994 and 2018 (of whom 47.9 % were men) were included in the study. data were collected from medical records. medcalc statistical software version 17.8.2 was used for the statistical analysis, with significance set at α = 0.05. results: forty-three of 144 mm patients (29.9 %) were treated with hd and/or pe. male patients required hd or pe more often than female patients (62.8 % vs 37.2 %, p = 0.02). patients who did not require hd or pe were significantly older at the time of their death than the patients treated with hd or pe [75 (interquartile range, iqr, 72 – 77) vs 72 (iqr 66 – 75) years; p = 0.009, mann-whitney test]. among all patients who required acute or chronic hd, pe or a combination of the treatments, the longest life span was found in 17 patients who were treated with chronic hd (median 12 months, iqr 8 – 58). conclusion: kidney failure requiring hd or pe in mm was associated with a significantly shorter life span in comparison with other mm patients. chronic hd patients had the longest survival among patients who required acute or chronic hd, pe or a combination of the treatments. in general, mm patients in need for hd and/or pe had poor survival. (smajić* p, schönberger e, periša v, sinčić petričević j, zibar l, kralik k. survival of multiple myeloma patients undergoing dialysis or plasma exchange a single centre’s 25-year experience. seemedj 2020; 4(1); 25-31) seemedj 2020, vol 4, no. 1 survival of multiple myeloma patients undergoing dialysis 26 southeastern european medical journal, 2020; 4(1) introduction multiple myeloma (mm) represents malignant plasma cell proliferation. tumours, their products and the reactions of the host that they produce lead to a number of dysfunctional organ conditions and symptoms, such as bonerelated pain or fractures, kidney failure, susceptibility to infections, anaemia, hypercalcemia and sometimes clotting irregularities, neurological symptoms and symptoms of hyperviscosity (1). kidney failure occurs in nearly 25 % of the patients, while in more than half of mm patients, there is some kidney pathology. hypercalcemia, deposition of amyloid in glomeruli, hyperuricemia, recurrent infections and kidney infiltration with tumour cells contribute to kidney disorders (1). in most cases, kidney damage is associated with immunoglobulin (ig) and it includes the following conditions: light chain cast nephropathy (lccn), also known as myeloma kidney, monoclonal immunoglobulin deposit disease and light chain amyloidosis (2). an increased quantity of light chains overloads tubular cells, which causes tubular damage, resulting in direct toxic effects of the light chain or indirect release of cell lysosomal enzymes. it can also cause fanconi syndrome by the formation of crystalline inclusions in the proximal tubules, resulting in a high loss of glucose and amino acids as well as irregularities of acidification and urea concentration (1, 2). plasma exchange (pe) has been suggested as a treatment option for lccn in order to prevent kidney failure. some retrospective studies have shown that pe can prevent acute kidney injury (aki), which can verge into chronic kidney disease (ckd) and the need for dialysis (3), while other studies have considered pe as a treatment that does not have any effect on overall survival or need for haemodialysis (2, 4). other treatment options for patients with mm and renal failure include bortezomib-based therapies or the use of high cut-off dialysis filters to remove free light chains (5). the administration of peritoneal dialysis and kidney transplant in mm patients with kidney failure are controversial treatment procedures because of their low rate of survival and poor tolerance of therapy (6). at the university hospital centre osijek, a proportion of mm patients were treated with acute or chronic hd, pe or a combination of the treatments. the focus of the study was to analyse the outcome in mm patients who required hd or pe. subjects and methods the retrospective study included all 144 patients treated at the university hospital centre osijek for mm during the period between 1994 and 2018. available patients’ data were collected from medical records at the department of hematology, the department of nephrology and from the register of deaths at the clinical department of pathology and forensic medicine of the university hospital centre osijek. the study was conducted between june 2017 and february 2018. mm diagnosis was confirmed if the bone marrow analysis showed plasmacytosis (> 30 %) or if plasmacytosis > 10 % was accompanied by monoclonal paraprotein in serum and/or urine and osteolytic lesions or light chains in serum were present. criteria for the start of hd treatment were aki or clinical and laboratory signs of ckd that required substitution of renal function. pe was performed for hyperviscosity to remove the light chains. statistical methods category data were represented by absolute and relative frequencies. the numerical data were described as median, minimum, maximum and interquartile range (iqr). differences in nominal variables were tested by fisher’s exact test. differences in numeric variables, due to deviations from normal distribution, were tested by mann-whitney's u test. all p values were double-sided. the level of significance was set at α = 0.05. medcalc statistical software version 17.8.2 was used for the statistical analysis (medcalc software bvba, ostend, belgium; http://www.medcalc.org; 2017). seemedj 2020, vol 4, no. 1 survival of multiple myeloma patients undergoing dialysis 27 southeastern european medical journal, 2020; 4(1) results the study included 144 mm patients, 43 (29.9 %) of whom required hd or pe. male patients needed hd or pe significantly more often (27 of 69, 62.8 %) than female patients (16 of 75, 37.2 %), p = 0.02 (fisher's exact test). up to the time of the study, 95 mm patients died (66 %), and at a significantly higher rate if a patient needed hd or pe (34 of 43, 79.1 %) than those who were not candidates for the treatments (61 of 101, 60.4 %), p = 0.03 (fisher’s exact test) (table 1). table 1. characteristics of patients (n = 144) with multiple myeloma (mm) with regard to haemodialysis (hd) or plasma exchange (pe) treatment and overall survival at the time of study number of (%) patients p* mm patients who required hd or pe treatment (n = 43, 29.9 %) mm patients who did not required hd or pe treatment (n = 101, 70.1 %) total gender male 27 (62.8) 42 (41.6) 69 (47.9) 0.02 female 16 (37.2) 59 (58.4) 75 (52.1) overall survival alive 9 (20.9) 40 (39.6) 49 (34) 0.03 deceased 34 (79.1) 61 (60.4) 95 (66) total 43 (100) 101 (100) 144 (100) *fisher’s exact test among 43 mm patients who required hd or pe treatment, 12 (28 %) were treated only with acute hd, 11 (26 %) were treated only with chronic hd and 8 (19 %) only with pe. seven (16 %) mm patients were treated with both acute hd and pe, 1 (2 %) mm patient was treated with both chronic hd and pe and 1 (2 %) with both acute and chronic hd. finally, there were 3 (7 %) mm patients treated with acute hd, chronic hd and pe (table 2).. table 2. distribution of patients according to the type of treatment acute or chronic haemodialysis (ahd or chd) or plasma exchange (pe) (n = 43) treatment n % ahd 12 28 chd 11 26 pe 8 19 ahd+pe 7 16 chd+pe 1 2 ahd+chd 1 2 ahd+chd+pe 3 7 total 43 100 seemedj 2020, vol 4, no. 1 survival of multiple myeloma patients undergoing dialysis 28 southeastern european medical journal, 2020; 4(1) among 43 (29.9 %) mm patients who required hd or pe treatment, a total of 16 (37.2 %) patients were treated with chronic hd, including those treated with acute hd or pe in combination with chronic hd. their median survival time was 11 months (iqr 8 – 58). a total of 19 (44.2 %) mm patients were treated with acute hd, including those treated with chronic hd or pe in combination with acute hd and their median survival time was 2 months (iqr 1 – 5). eight (18.6 %) mm patients were treated only with pe and their median survival time was 1 month (iqr 0 – 20). there was a significant difference in survival time among the examined groups (p = 0.04) (figure 1).. figure 1. kaplanmeier plot of survival time (months) between the groups of patients who required haemodialysis (hd) or plasma exchange (pe) treatment. chd includes multiple myeloma (mm) patients treated with chronic hd, including those treated with acute hd or pe in combination with chronic hd. their median survival time was 11 months (iqr 8 – 58). ahd includes mm patients who were treated only with acute hd and those treated with a combination of acute hd and pe. their median survival time was 2 months (iqr 1 – 5). pe encompasses mm patients who were treated only with pe and their median survival time was 1 month (iqr 0 – 20). there was a significant difference in survival time among the examined groups (p = 0.04). survival time was calculated from the start of the treatment until the patient’s death. a total of 35 (81.4 %) mm patients were treated with hd, either only hd or in combination with pe, and their median survival time was 7 months (iqr 2 – 12). there were 8 (18.6 %) mm patients treated only with pe and their median survival time was 1 month (iqr 0 – 20). there was no significant difference in survival time between those who were treated with hd (including patients treated only with hd or with a combination of hd and pe) and those treated only with pe (p = 0.39) (figure 2).. p seemedj 2020, vol 4, no. 1 survival of multiple myeloma patients undergoing dialysis 29 southeastern european medical journal, 2020; 4(1) figure 2. kaplan-meier plot of survival (months) between the groups of patients who were treated only with plasma exchange (pe) and those treated with haemodialysis (hd) (includes patients treated only with hd or with a combination of hd and pe). the median survival time of multiple myeloma (mm) patients treated with pe was 1 month (iqr 0 – 20). the median survival time of the other group was 7 months (iqr 2 – 12). there was no significant difference in survival time between these two groups (p = 0.39). discussion impairment of kidney function in mm patients requiring dialysis or pe was associated with a shorter life span than in mm patients without such treatments. patients who had no need for treatment with hd or pe were older at the time of death than the patients who needed such treatment, as we had assumed. the need for dialysis or pe was accompanied by a poor outcome in terms of life span in general. at the moment of our research, the outcome of the treatment was negative (death of the patient) in 95 (66 %) cases; significantly, 34 (79.1 %) of these patients needed pe and/or hd. ckd often appears in mm patients. despite aggressive treatment, 65 % of mm patients with kidney damage will reach the end-stage renal disease within three months of their mm diagnosis (7). studies have shown that mortality and morbidity are more common among patients who need renal replacement therapy (rrt) than among those with regular kidney function (8). these findings correspond with our results. some research has demonstrated that patients with mm and impairment of kidney function can be treated either with hd or pe and that these procedures are equally effective (9). on the other hand, one retrospective study suggested that pe might be helpful in preventing the initiation or continuation of dialysis in patients with rapidly progressive renal failure which occurred due to mm (10). more recent studies have suggested that pe in combination with chemotherapy, especially bortezomib, leads to renal improvement (11, 12). in this research, among the patients who needed hd or pe, the 17 patients who were treated with chronic hd lived the longest. studies conducted at different centres found contrasting results. therefore, in the future, research on this issue should be conducted on a larger number of subjects to get the most accurate results. seemedj 2020, vol 4, no. 1 survival of multiple myeloma patients undergoing dialysis 30 southeastern european medical journal, 2020; 4(1) our research was a retrospective epidemiologic study. it should be noted that there were some limitations of this study due to data scarcity for the group without hd or pe treatment, which can account for some of the differences between our research and studies conducted by other researchers. there were missing facts about the time when the diagnoses of mm were confirmed, stage and type of mm, presence of kidney failure in those without non-conservative treatment, presence of other complications and methods of pharmacological treatment. however, the results of our research are important in analysing the outcomes of treatment at one centre and they show the negative association of kidney failure as well as other methods of treatment with nonconservative procedures on lifespan and death outcome. it is also important to emphasize that there has been no similar research published by other centres. conclusion our study has shown that the requirement of chronic hd is related to a better outcome than that of acute hd or pe when it comes to length of life. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. . references 1. ivaničević ž, rumboldt z, bergovec m, silobrčić v, bruketa d, editors. harrison: principi interne medicine. 1st croatian ed. split: placebo; 1997. 2. vakiti a, padala sa, mewawalla p. myeloma kidney. [updated 2020 jan 25]. in: statpearls [internet]. treasure island (fl): statpearls publishing; 2020 jan-. available on: https://www.ncbi.nlm.nih.gov/books/nbk499 952/?report=reader#_nbk499952_pubdet_ (accessed 3 march 2020) 3. goldschmidt h, lannert h, bommer j, ho ad. multiple myeloma and renal failure. nephrol dial transplant 2000; 15:301-304. doi: https://doi.org/10.1093/ndt/15.3.301 4. clark wf, stewart k, rock ga, sternbach m, sutton dm, barrett bj et al.; canadian apheresis group. plasma exchange when myeloma presents as acute renal failure: a randomized, controlled trial. ann intern med 2005; 143:777-784. 5. moreau p, san miguel j, sonneveld p, mateos mv, zamagni e, avet-loiseau h, hajek r, dimopoulos ma, ludwig h , einsele h, zweegman s, facon t, cavo m, terpos e, goldschmidt h, attal m, buske c. multiple myeloma: esmo clinical practice guidelines for diagnosis, treatment and follow-up. ann oncol 2017; 28 (suppl 4): iv52–iv61. 6. reyes gm, valera a, frutos ma, ramos b, ordóñez v, lópez de novales e. supervivencia de pacientes con mieloma tratados con diálisis [survival of myeloma patients treated with dialysis]. nefrologia 2003; 23(2):131-136. 7. katagiri d, noiri e, hinoshita f. multiple myeloma and kidney disease. scientific world journal 2013; 487285:9. doi: https://www.hindawi.com/journals/tswj/2013 /487285/ 8. ganeval d, rabian c, guérin v, pertuiset n, landais p, jungers p. treatment of multiple myeloma with renal involvement. adv nephrol necker hosp 1992; 21:347–370. 9. clark ad, shetty a, soutar r. renal failure and multiple myeloma: pathogenesis and treatment of renal failure and management of underlying myeloma. blood rev 1999;13(2):79– 90. 10. moist l, nesrallah g, kortas c, espirtu e, ostbye t, clark w.f. plasma exchange in rapidly progressive renal failure due to multiple myeloma. am j nephrol 1999; 19:45–50. seemedj 2020, vol 4, no. 1 stigmatization of patients with viral infections and mass psychogenic illness 31 southeastern european medical journal, 2020; 4(1) 11. premuzic v, batinic j, roncevic p, basicjukic n, nemet d, jelakovic b. role of plasmapheresis in the management of acute kidney injury in patients with multiple myeloma: should we abandon it? ther apher dial 2018; 22(1):79-86. 12. burnette bl, leung n, rajkumar sv. renal improvement in myeloma with bortezomib plus plasma exchange. n engl j med 2011; 364:2365– 2366. seemedj 2020, vol 4, no. 1 foetal sex prediction 96 southeastern european medical journal, 2020; 4(1) original article he or she, what will it be: old wivesʹ tales and foetal sex prediction 1 darjan kardum* 1, željka kardum 2, tihana nađ 1, andrijana müller 3 1 neonatal intensive care unit, department of paediatrics, university hospital centre osijek, faculty of medicine osijek, josip juraj strossmayer university of osijek, croatia 2 department of rheumatology, clinical immunology and allergology, university hospital centre osijek, faculty of medicine osijek, josip juraj strossmayer university of osijek, croatia 3 department of gynaecology and obstetrics, university hospital centre osijek, faculty of medicine osijek, josip juraj strossmayer university of osijek, croatia *corresponding author: darjan kardum, darjankardum@gmail.co received: feb 22, 2020; revised version accepted: apr 3, 2020; published: apr 27, 2020 keywords: folklore, parents, pregnancy, maternal nausea abstract aim: a myriad of myths surround pregnancy, especially regarding the prediction of the sex of the infant. some of these myths and old wives' tales are, to this day, widespread among expectant parents. the objective of this study was to examine whether common pregnancy-related statements regarding foetal sex prediction vary between mothers of female and male infants. methods: the questionnaire-based study was conducted from september 2017 to september 2018 at a well-baby nursery. participants were mothers of infants (n = 350) admitted to the well-baby nursery with a singleton pregnancy and at > 36 weeks of gestation at birth. results: we investigated a number of statements regarding foetal sex prediction. with the exception of one statement, there were no other differences between mothers of male and female infants. pregnancy with a male foetus is associated with glossier hair during pregnancy. women with female newborns reported glossier hair during pregnancy in 39.1% of cases, while women with male newborns reported the same in 45.0% of cases (p = 0.04). conclusion: old wives' tales regarding sex prediction of the infant during pregnancy remain myths for a reason, with the possible exception of one statement regarding glossier hair and pregnancy with a male foetus. (kardum* d, kardum ž, nađ t, müller a. he or she, what will it be: old wivesʹ tales and foetal sex prediction. seemedj 2020; 4(1); 96-101) seemedj 2020, vol 4, no. 1 foetal sex prediction 97 southeastern european medical journal, 2020; 4(1) introduction since the beginning of recorded history, people have been continuously fascinated with pregnancy. pregnancy is possibly associated with more myths and folklore tales than any other human condition and this fascination exists even today. a myriad of myths surround pregnancy, especially regarding prediction of the sex of the infant. since ancient times, several tell-tale signs have been associated with the birth of a male or a female child. hippocrates states that “women beget females if they have rough spots on the face; those who keep a good complexion beget males” (1). some of these myths and old wives' tales are, to this day, widespread among expectant parents. the aim of this study was to examine whether common and widespread pregnancy-related statements regarding foetal sex prediction vary between mothers of female and male infants. methods to investigate the popular myths regarding foetal sex prediction in pregnancy, we surveyed 350 mothers of singleton newborn infants with gestational age > 36 weeks, who were admitted to a well-baby nursery at the department of gynaecology and obstetrics at university hospital centre osijek, croatia from september 2017 to september 2018. the study protocol was approved by the uhc osijek ethics committee. a questionnaire was administered to mothers of newborn infants and data on eleven common pregnancy myths were collected. in addition, they were asked two questions aimed to determine the link between maternal heartburn intensity and their perception of the infant's hair density, and one question regarding the maternal sentiment about the sex of the infants at the time they found out that they were pregnant. statistical anaylsis data were described by using descriptive statistical methods. the chi-squared test was used to analyse the differences between proportions compare samples. all p values are two-sided. level of significance is set at alpha = 0.05. statistical analysis was performed by using the statistical program medcalc statistical software version 18.9 (medcalc software bvba, ostend, belgium; http://www.medcalc.org; 2018). results a total of 350 women responded to the questionnaire: 179 of them were mothers of female infants and 171 of male infants. the results are shown in table 1. seemedj 2020, vol 4, no. 1 foetal sex prediction 98 southeastern european medical journal, 2020; 4(1) table 1. common pregnancy myths *chi-square test statement number (%) according to sex of the infant p female (n=179) male (n=171) total during the pregnancy, i carried the baby: “high” 103 (57.5) 86 (50.3) 189 (54) 0.17 “low” 76 (42.5) 85 (49.7) 161 (46) during the pregnancy, my facial complexion was: poorer than before 39 (21.8) 27 (15.8) 66 (18.9) 0.15 unchanged or better than before 140 (78.2) 144 (84.2) 284 (81.1) during the pregnancy, i had: mild or no morning sickness 124 (69.3) 120 (70.2) 244 (69.7) 0.85 strong morning sickness 55 (30.7) 51 (29.8) 106 (30.3) during the pregnancy, i had: mildly or moderately increased appetite 115 (64.2) 125 (73.1) 240 (68.6) 0.08 markedly increased appetite 64 (35.8) 46 (26.9) 110 (31.4) during the pregnancy, i craved mostly on: sweet foods 87 (48.6) 85 (49.7) 172 (49.1) 0.84 salty foods 92 (51.4) 86 (50.3) 178 (50.9) at the end of the pregnancy, my belly had the shape of: a watermelon 92 (51.4) 80 (46.8) 172 (49.1) 0.39 a soccer ball 87 (48.6) 91 (53.2) 178 (50.9) during the pregnancy, i had wild mood swings, i was occasionally sad and of lower mood: no 83 (46.4) 78 (45.6) 161 (46) 0.89 yes 96 (53.6) 93 (54.4) 189 (54) at the end of the pregnancy, i mostly woke up on my: right side of the body 71 (39.7) 56 (32.7) 127 (36.3) 0.18 left side of the body 108 (60.3) 115 (67.3) 223 (63.7) during the pregnancy, my hair was: the same as before the pregnancy 118 (65.9) 94 (55) 212 (60.6) 0.04 glossier than before 61 (34.1) 77 (45) 138 (39.4) during the pregnancy, i gained more weight: on the central part of my body 104 (58.1) 93 (54.4) 197 (56.3) 0.52 on the hips and backside 75 (41.9) 78 (45.6) 153 (43.7) during the pregnancy, my partner: gained weight 63 (35.2) 57 (33.3) 120 (34.3) 0.71 stayed the same weight 116 (64.8) 114 (66.7) 230 (65.7) total 179 (100) 171 (100) 350 (100) seemedj 2020, vol 4, no. 1 foetal sex prediction 99 southeastern european medical journal, 2020; 4(1) we found no statistical significance between 10 popular statements regarding sex prediction during pregnancy. the only exception was a myth that states that a pregnancy with a male foetus is associated with glossy hair during pregnancy. in the group of women with female foetuses, 39.1% of them reported glossier hair during pregnancy, while in the group of women with male foetuses, 45.0% of them reported the same. the difference is significant (p = 0.04). with regard to the correlation between hair volume and heartburn intensity, 205 women described that their newborn has “a lot of hair” and 145 women described that their newborn has “scarce or no hair”. mothers of infants with a lot of hair reported severe heartburn in 45.9% of cases and those with “scarce or no hair” reported severe heartburn in 34.5% of cases (p = 0.03). mothers accurately predicted the sex of the infant at the time they first found out that they were pregnant in 60% of the cases (57.5 % in cases of female pregnancy and 62.6% in cases of male pregnancy). discussion since the first artistic depictions of the human figure in history, a collection known as venus figurines, some of which are perceived as representations of pregnant women (2), we have been witnesses of the continuing fascination with pregnancy. this interest in pregnancy has not diminished even today, which is clearly reflected in the fact that a google search of the term “foetal sex prediction myths” yields more than 14 million search results. there have been several studies conducted in an attempt to validate some of the folklore tales and myths surrounding pregnancy. hsu et al (3) found that women with severe hyperemesis gravidarum were more likely to have female foetuses. the authors propose a mechanism of action in which hyperemesis gravidarum is caused by the female foetal effect on hcg concentrations. this finding was first reported by schoeneck et al (4). they found that the concentration of gonadotropic hormone in the urine of pregnant patients who present symptoms of nausea and vomiting was increased, compared to pregnant patients who are free from these symptoms. later studies confirmed these findings (5-7). another common myth is that severe morning sickness is associated with female sex of the foetus. in our study, we found that severe morning sickness was found in 30.7% of women with female foetuses and in 29.8% of women with male foetuses. the difference was not statistically significant, but some studies have so far reported that women presenting with morning sickness in the first trimester are more likely to give birth to a female infant (8, 9). it is a common myth that cravings for sweet food are associated with pregnancies with female foetuses. a similar number of pregnant women with both male (49.7%) and female (48.6%) infants craved sweet food. this difference is not significant. the shape of the belly, carrying the baby “low” or “high” and distribution of additional weight gain during pregnancy were not statistically significant in pregnancies with female and male foetuses. perry et al (10) investigated some beliefs associated with predicting foetal sex (e.g., whether a woman is carrying her foetus in front or across the hips, prevalence of morning sickness) and concluded that these were ineffective. ostler et al. (11) investigated the predictive value of three common pregnancy myths (foetal heart rate test, the chinese calendar test, and the draino test) and found that these tests had no value in terms of sex prediction. among other myths, we investigated a common myth stating that if the partner of an expectant mother gained weight (the so-called “chubby hubby” test), that this is indicative of a pregnancy with a female foetus. interestingly, 34.3% of male partners of expectant mothers gained weight. however, no statistical difference was found in mothers of male or female infants. of the 11 investigated myths, the only myth that produced a significant difference between mothers of male and female infants is the one stating that mothers of male infants report higher incidences of glossy hair during seemedj 2020, vol 4, no. 1 foetal sex prediction 100 southeastern european medical journal, 2020; 4(1) pregnancy (45%), compared to mothers of female infants (34.1%). it is challenging to propose a physiological process, but some researchers have suggested that testosterone has a positive anabolic effect on hair growth (12), and some researchers reported higher maternal peripheral testosterone levels during the first half of the pregnancy in mothers with male foetuses (13). regarding the connection between infants’ hair density and incidence and duration of morning sickness, some interesting results were obtained through our study. we found that women who describe their infant as having “a lot of hair” reported severe heartburn in 45.9% of cases and those with “scarce or no hair” reported severe heartburn in 34.5% of cases. the difference is statistically significant (p = 0.03). surprisingly, this finding is consistent with costigan et al (14) who reported a simple linear relationship between maternal heartburn severity and infants’ hair volume. in addition, they have found that women who reported moderate or severe heartburn gave birth to babies with average or above average amounts of hair. conversely, most women reporting no heartburn had babies with less than average or no hair. the authors hypothesize that individual variations in pregnancy hormone levels that have been implicated in the relaxation of the lower oesophageal sphincter and resultant reflux are also independently associated with hair growth of the foetus (14). we found that the most common pregnancy myths had no empirically validated results. however, an interesting relationship was found between maternal heartburn intensity and maternal perception of the infant’s hair density. also, we found statistically significant results related to the myth stating that mothers with male infants reported glossier hair during pregnancy. the main limitation of this study is that the mothers’ perception related to some of the statements that were made could be attributed to the fact that these statements were examined after the sex of the newborn was already known. consequently, in retrospect, some claims could be reinforced and others discarded (e.g. heartburn intensity, shape of the belly, etc.). we hope that the results of this study will not diminish the enthusiasm of expectant parents related to common pregnancy myths regarding sex prediction of the foetus, especially since pregnancy folklore tales and myths play an important part in the lives of expectant parents. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. . references 1. mccartney es. sex determination and sex control in antiquity. am j philol. 1922; 43(1):62. 2. dixson af, dixson bj. venus figurines of the european paleolithic: symbols of fertility or attractiveness? journal of anthropology. 2011; 2011:1–11. 3. hsu c, witter f. fetal sex and severe hyperemesis gravidarum. int j gynaecol obstet. 1993; 40(1):63–4. 4. schoeneck fj. gonadotropic hormone concentration in emesis gravidarum. am j obstet gynecol. 1942;43(2):308–12. 5. goodwin tm, hershman jm, cole l. increased concentration of the free β-subunit of human chorionic gonadotropin in hyperemesis gravidarum. acta obstet gynecol scand. 1994; 73(10):770–2. 6. goodwin tm, montoro m, mestman jh, pekary ae, hershman jm. the role of chorionic gonadotropin in transient hyperthyroidism of hyperemesis gravidarum. j clin endocrinol metab. 1992; 75(5):1333-7. 7. kauppila a, huhtaniemi i, ylikorkala o. raised serum human chorionic gonadotrophin seemedj 2020, vol 4, no. 1 stigmatization of patients with viral infections and mass psychogenic illness 101 southeastern european medical journal, 2020; 4(1) concentrations in hyperemesis gravidarum. bmj. 1979; 1(6179):1670–1. 8. galanakis e. sickness and sex of child. lancet. 2000; 355(9205):756. 9. askling j, erlandsson g, kaijser m, akre o, ekbom a. sickness in pregnancy and sex of child. lancet. 1999; 354(9195):2053. 10. perry df, dipietro j, costigan k. are women carrying "basketballs" really having boys? testing pregnancy folklore. birth. 1999; 26(3):172-177. 11. ostler s, sun a. fetal sex determination: the predictive value of 3 common myths. cmaj. 1999; 161(12):1525-6. 12. glaser rl, dimitrakakis c, messenger ag. improvement in scalp hair growth in androgendeficient women treated with testosterone: a questionnaire study. br j dermatol. 2012; 166(2):274-8. 13. klinga k, bek e, runnebaum b. maternal peripheral testosterone levels during the first half of pregnancy. am j obstet gynecol. 1978; 131(1):60-62. 14. costigan ka, sipsma hl, dipietro ja. pregnancy folklore revisited: the case of heartburn and hair. birth. 2006; 33(4):311-314. seemedj 2022, vol 6, no 2 somatic illnesses and mental disorders treatment 54 southeastern european medical journal, 2022; 6(2) original article comorbidity of somatic illnesses on people with treated mental disorders – a new challenge in medicine 1 romana marušić 1,2, adriana levaković 2, dunja degmečić 2,3, tatjana bačun 2,4* 1 department of internal medicine, national memorial hospital vukovar, croatia 2 faculty of medicine, j.j. strossmayer university, osijek, croatia 3 department of psychiatry, university hospital centre, osijek, croatia 4 division of endocrinology, department of internal medicine, university hospital centre, osijek; croatia *corresponding author: tatjana bačun, tbacun@gmail.com received: apr 12, 2022; revised version accepted: oct 20, 2022; published: nov 28, 2022 keywords: chronic diseases, comorbidity, mental disorders abstract aim. comorbidities pose a major challenge for 21st century medicine. the mutual pathophysiological effect of one disease on another can significantly affect their course and prognosis. the aims of this study were to examine the frequency of comorbidities and the most common psychiatric and somatic comorbidities and to determine the difference in the incidence of certain diseases by gender and age. methods. data were recorded in several groups: demographic characteristics, psychiatric and somatic diagnoses classified according to gender, age, and the legally determined ability to work, and correlations of somatic and psychiatric diagnoses. results. the most common psychiatric diagnoses in men were post-traumatic stress disorder (ptsd) (25%) and alcoholism (23%), while in women these were recurrent depressive disorder (19%) and psychosis (10%). a statistically significant difference was found in the incidence of alcoholism and ptsd, which are more common in men than in women. the most common somatic diseases in both sexes were arterial hypertension (m = 33%, f = 46%) and diabetes mellitus (m = 18%, f = 32%). statistically significant differences were found in the frequency of hypertension (p = 0.03) and epilepsy (p = 0.002), which are more common in men. the ratio alcoholism-hypertension (p = 0.03), alcoholism-diabetes (p < 0.0001), alcoholism-copd (p < 0.001) was statistically significant. conclusion. it is extremely important to improve the multidisciplinary approach and cooperation in treatment in order to reduce the number of hospitalizations, emergency interventions and suicides and to improve the patients’ quality of life and life expectancy. (marušić r, levaković a, degmečić d, bačun t*. comorbidity of somatic illnesses on people with treated mental disorders – a new challenge in medicine. seemedj 2022; 6(2); 54-66) seemedj 2022, vol 6, no 2 somatic illnesses and mental disorders treatment 55 southeastern european medical journal, 2022; 6(2) introduction comorbidity indicates the presence of two or more different diseases or disorders simultaneously and poses a major challenge for 21st century medicine (1). people with a chronic physical illness are 1.5 to 2 times more likely to develop a mental disorder (2). it is estimated that 25% of the general population has some form of mental disorder. of these, as many as 68% have one or more physical comorbidities (3). due to the extended average lifespan, the prevalence of multimorbidity is increasing; in australia, it is 75% for people aged 65 to 74 and over 80% for people aged 75 and above. a study conducted in ontario showed a multimorbidity of 7 to 35 % in people between the ages of 18 and 65 (4). in croatia, the prevalence of multimorbidity is 79.8 % for people over 65 (5). mental disorders are a major public health problem that has a significant impact on the health of people with chronic diseases and can change the course of their illness and their prognosis. depression is present in 40% of people with hypertension; the prevalence is 36.6% in men and as much as 63.4% in women. in somatically healthy individuals with depressive disorder, the risk of coronary heart disease is increased 1.5to 2-fold, while in individuals with coronary heart disease, the risk of myocardial infarction is increased 1.5to 4.5-fold (2). some studies show that the prevalence of depressive disorders in people with diabetes ranges from 8 to 15% (6). only 25% to 50% of people with diabetes who suffer from depression get diagnosed and treated (7). the risk of complications of the disease is increased as much as 4 times due to the reduced ability to regulate glucose metabolism (1, 8). other studies show that as many as 47.6% of young people with insulin-dependent diabetes develop a mental disorder after ten years, most often one year after diagnosis (2). the cost of hospital treatment of patients with comorbid depression was increased 1.5-fold compared with other patients. the positive correlation of psychological comorbidities with the length of hospital stays, doctor visits, and longer sick leave has also been proven, which leads to a reduced quality of life and higher treatment costs (2). one study showed that every fifth person in physical rehabilitation suffers from a comorbid mental disorder. this has an adverse effect on the outcome of rehabilitation due to the patient’s reduced motivation, cooperation, and active participation in the rehabilitation process (9). due to the high incidence of comorbidities of physical and mental illnesses, it is important to conduct preventive examinations, personalized pharmacotherapy and psychotherapy, and educational programs for medical professionals. an integrative approach and timely recognition and treatment can reduce mortality, morbidity, and overall treatment costs (10). the aims of our research were to examine the frequency of comorbidities and the most common psychiatric and somatic comorbidities and to determine the difference in the incidence of certain diseases by gender and age. patients and methods patients and study design the research was organized as a cross-sectional study with historical data. it was approved by the ethics committee of the faculty of medicine osijek, josip juraj strossmayer university in osijek. the study was conducted on 137 subjects who were hospitalized at the clinic for psychiatry of the clinical hospital center osijek. data were collected from the hospital information system of the clinical hospital center osijek over a period of one year. inclusion criterion was diagnosis of any somatic illness in psychiatric patients. methods data were categorized in several groups: demographic characteristics of respondents, psychiatric and somatic diagnoses classified seemedj 2022, vol 6, no 2 somatic illnesses and mental disorders treatment 56 southeastern european medical journal, 2022; 6(2) according to gender, age, and the legally determined ability to work (up to 65 years), and correlations of somatic and psychiatric diagnoses. to examine the correlation between somatic and psychiatric diagnoses, only diagnoses for which frequency was higher than 3% (n ≥ 7 for psychiatric, n ≥ 5 for somatic diagnoses) in total population and somatic diagnoses that are applicable in both genders came into consideration. statistical analysis r software was used to perform statistical analysis of the collected data. descriptive data are expressed in frequency and share for nominal variables and arithmetic and standard deviation for numerical variables that have a normal distribution. the normality of distribution was examined using the kolmogorov-smirnov test. differences of category variables were tested by binomial, χ2, and fisher’s exact test, and the degree of correlation was examined by the φ test. differences of numerical variables with normal distribution were tested by student’s t-test. the level of statistical significance was set at p < 0.05. results the study included 137 respondents, of whom 95 (70%) were men and 42 (30%) were women. the mean age of men was 55.9 ± 12.1 (22 to 87) and women 56.9 ± 13.5 (16 to 80). of the 137 respondents, 2% were aged between 18 and 30, 76% were between 30 and 65, and 23% were over 65. a statistically significant difference was found in the number of respondents by sex, in the group aged 35 to 65 years (p < 0.0001). respondents were divided according to employment into employed, unemployed, and retired ones: a statistically significant difference was found between retired men and women (p < 0.0001) (table 1). table 1. demographic characteristics of respondents male female total p age 55.9+12,2 56.9+13,5 0.68 age category 16-30 2(20%) 1(2%) 3(2%) 0.56 30-65 73(77%) 30(71%) 103(76%) < 0.0001 65 + 20(20%) 11(26%) 31(23%) 0.15 employment status employed 15(16%) 3(7%) 18(13%) < 0.05 unemployed 22(23%) 16(39%) 38(28%) 0.41 retired 57(60%) 22(54%) 79(59%) < 0.0001 the most common psychiatric diagnoses in men were post-traumatic stress disorder (ptsd) (25%), alcoholism (23%), recurrent depressive disorder (13%), while in women the most common ones were recurrent depressive disorder (19%), psychosis (10%), alcoholism, ptsd, and anxiety-depressive disorder (7%). a statistically significant difference was found in the incidence of alcoholism and ptsd, which were more common in men than in women (table 2). seemedj 2022, vol 6, no 2 somatic illnesses and mental disorders treatment 57 southeastern european medical journal, 2022; 6(2) table 2. psychiatric diagnoses of respondents male female total p alcoholism 33(23%) 4(7%) 37(18%) < 0.0001 recurrent depressive disorder 18(13% 11(19%) 29(14%) 0.26 ptsd* 35(25%) 4(7%) 39(19%) < 0.0001 psychoorganic syndrome 7(5%) 2(3%) 9(4%) 0.18 anxiety-depressive disorder 3(2%) 4(7%) 7(3%) 1 psychosis 8(6%) 6(10%) 14(7%) 0.79 another inorganic psychotic disorder 1(1%) 0(0%) 1(0%) pervasive developmental disorder 0(0%) 1(2%) 1(0%) mild mental retardation 4(3%) 1(2%) 5(2%) 0,27 delirium tremens 3(2%) 0(0%) 3(1%) organic insane disorder 1(1%) 3(5%) 4(2%) 0,62 schizophrenia 6(4%) 3(5%) 9(4%) 0.5 borderline depressive decompensation 4(3%) 3(5%) 7(3%) 1 depressive disorder 10(7%) 3(5%) 13(6%) 0.09 bipolar affective disorder 2(1%) 4(7%) 6(3%) 0.68 crisis 1(1%) 4(7%) 5(2%) 0.37 ocd* 3(2%) 0(0%) 3(1%) schizoaffective disorder 2(1%) 3(5%) 5(2%) 1 organic emotionally labile disorder 0(0%) 3(5%) 3(1%) anorexia nervosa 1(1%) 0(0%) 1(0%) *ptsd – post-traumatic stress disorder, ocd – obsessive-compulsive disorder of the somatic diagnoses, the most common diagnoses in both sexes were arterial hypertension (m = 33%, f = 46%), diabetes mellitus (m = 18%, f = 32%) and epilepsy (m = 15%, f = 8%). statistically significant differences were found in the frequency of arterial hypertension (p = 0.03) and epilepsy (p = 0.002), which were more common in men (table 3). seemedj 2022, vol 6, no 2 somatic illnesses and mental disorders treatment 58 southeastern european medical journal, 2022; 6(2) table 3. somatic diagnoses of respondents male female total p arterial hypertension 41(33%) 23(46%) 64(36%) 0.03 diabetes mellitus 23(18%) 16(32%) 39(22%) 0.34 epilepsy 19(15%) 4(8%) 23(13%) 0.002 copd* 5(4%) 2(4%) 7(4%) 0.45 liver cirrhosis 1(1%) 0(0%) 1(1%) gastric ulcer 2(2%) 0(0%) 2(1%) pneumonia 2(2%) 0(0%) 2(1%) hashimoto's thyroiditis 0(0%) 1(2%) 1(1%) multiple sclerosis 1(1%) 0(0%) 1(1%) lumbosacral pain 2(2%) 0(0%) 2(1%) esophagitis 0(0%) 0(0%) 0(0%) chronic gastritis 5(4%) 0(0%) 5(3%) venous ulcer 1(1%) 0(0%) 1(1%) pulmonary embolism 0(0%) 1(2%) 1(1%) nhl* 1(1%) 0(0%) 1(1%) angina pectoris 3(2%) 0(0%) 3(2%) esophageal cancer 1(1%) 0(0%) 1(1%) psoriasis 4(3%) 0(0%) 4(2%) 1 prostate hyperplasia 5(4%) 0(0%) 5(3%) parkinson’s disease 3(2%) 0(0%) 3(2%) acute renal failure 1(1%) 0(0%) 1(1%) cystitis 1(1%) 0(0%) 1(1%) hepatitis 1(1%) 0(0%) 1(1%) cervicobrachial syndrome 0(0%) 1(2%) 1(1%) gerd* 1(1%) 0(0%) 1(1%) sle* 0(0%) 2(4%) 2(1%) urine retention 1(1%) 0(0%) 1(1%) hodgkin’s disease 1(1%) 0(0%) 1(1%) *copd – chronic obstructive pulmonary disease, nhl – non-hodgkin's lymphoma, gerd – gastroesophageal reflux disease, sle – systemic lupus erythematosus since we were interested in the frequency of psychiatric diagnoses among respondents, we divided them into groups based on their employment status and age. the most common illness-related psychiatric diagnoses among retired respondents were recurrent depressive disorder (31%), ptsd (26%), and alcoholism (12%). unemployed respondents were most often diagnosed with alcoholism (25%), recurrent depressive disorder (16%), and ptsd (14%). similar diagnoses were most common among employed respondents: alcoholism (33%) and seemedj 2022, vol 6, no 2 somatic illnesses and mental disorders treatment 59 southeastern european medical journal, 2022; 6(2) ptsd (11%). χ2 showed a significant statistical difference in the number of patients with ptsd and recurrent depressive disorder among retired, unemployed, and employed respondents under the age of 65 (table 4). table 4. psychiatric diagnoses according to the employment status retired unemployed employed > 65 < 65 > 65 < 65 p alcoholism 5(12%) 10(12%) 13(25%) 9(33%) 0.8 psychosis 5(12%) 0(0%) 4(8%) 2(7%) ptsd* 2(5%) 21(26%) 7(14%) 3(11%) 0.0004 schizophrenia 2(5%) 5(16%) 2(4%) 0(0%) borderline depressive decompensation 0(0%) 3(4%) 2(4%) 2(7%) 0.98 depressive disorder 5(12%) 2(2%) 5(10%) 2(7%) 0.63 bipolar affective disorder 0(0%) 4(5%) 1(2%) 1(4%) 0.5 mixed anxiety-depressive disorder 0(0%) 3(4%) 3(6%) 1(4%) 0.85 recurrent depressive disorder 5(12%) 25(31%) 8(16%) 2(7%) < 0.0001 organic emotionally labile disorder 3(7%) 0(0%) 0(0%) 0(0%) mild mental retardation 0(0%) 2(2%) 0(0%) 1(4%) crisis 1(2%) 1(1%) 1(2%) 287%) 0.96 psychoorganic syndrome 10(24%) 5(6%) 0(0%) 0(0%) organic delusional disorder 2(5%) 0(0%) 1(1%) 1(4%) anorexia nervosa 0(0%) 0(0%) 1(2%) 0(0%) schizoaffective disorder 0(0%) 0(0%) 4(8%) 0(0%) pervasive developmental disorder 0(0%) 0(0%) 0(0%) 0(0%) delirium tremens 1(2%) 0(0%) 0(0%) 1(4%) ocd* 0(0%) 0(0%) 0(0%) 0(0%) *ptsd – post-traumatic stress disorder, ocd – obsessive-compulsive disorder seemedj 2022, vol 6, no 2 somatic illnesses and mental disorders treatment 60 southeastern european medical journal, 2022; 6(2) figure 1. correlation of somatic and psychiatric diagnoses * ptsd – post-traumatic stress disorder, copd – chronic obstructive pulmonary disease with the aim of roughly determining the correlation of somatic and psychiatric diagnoses, a graph was made to reflect that correlation (figure 1). we took the most significant diagnoses from the graph and made a matrix in which we tested the statistical significance and correlation of the most common diagnoses: 1. psychiatric diagnoses: alcoholism, recurrent depressive disorder, ptsd, mixed anxietydepressive disorder, schizophrenia, depressive disorder 2. somatic diagnoses: hypertension, diabetes mellitus, epilepsy, copd, gastritis. the ratios alcoholism-hypertension (p = 0.03), alcoholism-diabetes mellitus (p < 0.0001), alcoholism-copd (p < 0.001) were statistically significant, which means that respondents treated for alcoholism had a lower risk for hypertension, diabetes, and copd, although the correlation was almost equal to zero (φ1 = 0.03, φ2 = 0, φ3 = 0.004) (table 5). 0 5 10 15 20 25 hypertension diabetes mellitus epilepsy copd gastritis seemedj 2022, vol 6, no 2 somatic illnesses and mental disorders treatment 61 southeastern european medical journal, 2022; 6(2) table 5. correlation of somatic and psychiatric disorders hypertension diabetes mellitus epilepsy copd gastritis yes no p φ yes no p φ yes no p φ yes no p φ yes no p yes 12 25 5 32 14 23 1 36 2 35 alcoholism 0.03 0.03 <0.000 1 0.18 0.18 <0.00 1 0.004 0.6 2 no 52 45 59 38 50 47 6 92 1 94 recurrent yes 15 14 8 21 1 28 3 26 2 27 depressive 0.678 1 0.03 0.03 0.17 0.3 disorder no 49 56 31 74 22 83 4 10 1 3 102 yes 20 19 12 27 5 34 3 36 2 37 ptsd 0.7 0.84 0.46 0.41 0.63 no 44 51 27 68 18 77 4 91 3 92 psychoorga nic yes 6 3 4 5 1 8 0 9 1 8 syndrome 0.31 0.45 1 0.29 no 58 67 35 90 22 103 7 11 8 4 121 mixed anxietyyes 5 2 4 3 0 7 1 6 0 7 depressive 0.26 0.19 0.32 disorder no 59 68 35 92 23 10 4 6 12 1 5 122 yes 5 4 3 6 1 8 0 9 0 9 psychosis 0.74 0.72 1 no 59 66 36 89 22 10 3 7 11 8 5 120 yes 2 6 3 5 1 7 1 7 0 8 schizophren ia 0.28 0.69 1 0.36 no 62 64 36 90 22 10 4 6 12 0 5 121 borderline yes 4 3 1 6 1 6 2 6 0 7 depressive 0.7 0.67 1 0.056 decompens ation no 60 67 38 89 22 10 5 5 12 1 5 122 depressive yes 8 4 2 10 2 10 1 11 0 12 disorder 0.23 0.5 1 0.49 no 56 66 37 85 21 10 1 6 11 6 5 117 bipolar yes 2 4 2 4 2 4 0 6 0 6 affective 0.69 1 0.27 disorder no 62 66 37 91 21 10 7 7 12 1 5 123 * ptsd – post-traumatic stress disorder, copd – chronic obstructive pulmonary disease discussion comorbidities of physical and mental disorders occur with high frequency, and they are most often present in people over 65 years of age. in this study, a statistically significant difference was found in the incidence of ptsd and alcoholism in men and women; they were significantly more common in men, while recurrent depressive disorder was more common in women. in our research, ptsd is almost four times more common in men. it is estimated that 3 to 6% of the population suffers from ptsd. seeing as some studies show that about 49.8% of people who were in the war develop ptsd, these findings can be related to the croatian war of independence in the early 1990s. ptsd was most often diagnosed in retired respondents under the age of 65 (26% of respondents), which seemedj 2022, vol 6, no 2 somatic illnesses and mental disorders treatment 62 southeastern european medical journal, 2022; 6(2) we can also attribute to the recent croatian war history. although several studies show increased incidence of somatic diseases in patients who suffer from ptsd, our study did not show a statistically significant correlation between ptsd and certain somatic diseases (11, 12, 13). one of the possible reasons could be a small number of participants in our study. ptsd is often associated with physical comorbidities ranging from nonspecific dizziness, tinnitus, and blurred vision to chronic pain, diabetes, cardiovascular, respiratory, and gastrointestinal diseases. there is also increased risk of cancer, arthritis, autoimmune and inflammatory diseases. in our study, the most common somatic disease in patients with ptsd was hypertension. a high ratio of patients with ptsd have unhealthy lifestyles and habits such as heavy smoking, low physical activity, and obesity, which lead to development of vascular, degenerative, and other types of somatic disorders (14). as many as 80% of people have at least one mental disorder with ptsd, the most common being depressive disorder, generalized anxiety disorder, and addictive disorder. due to non-cooperation, the frequency of hospitalizations and relapses is high (15). therefore, it is important to identify individuals suffering from this disease, encourage them to cope with the problem, and take care of their mental and physical health to avoid further consequences and comorbidities (16). in our study, the most common mental disorder in women was recurrent depressive disorder (19%). the most common comorbidity with a recurrent depressive disorder was hypertension (4). studies have shown that individuals with depression are more likely to develop hypertension, strokes, and ischemic heart disease. there is a pathophysiological connection between depression and hypertension because both disorders are characterized by increased sympathetic tone and increased secretion of adrenocorticotropic hormone and cortisol. moreover, depressed patients may have difficulty adhering to their therapy regimen, resulting in poor blood pressure control (17). the prevalence of depression is 10 to 15%, with over 350 million people currently affected. risk factors for developing depression disorder include stressful events, genetic predisposition, disability, illness, previous treatment for depression disorder and sleep problems. somatic chronic diseases increase the risk of developing depression due to reduced quality of life, difficulty coping with diagnosis, pain, and rejection of the environment (4). also, the prevalence of depression in people who suffer from at least one chronic illness is 9.3 to 23% and differs greatly from people who do not suffer from any chronic disorder (3.2 %). increased prevalence of depressive disorders has been noticed in people suffering from cardiovascular diseases (17-27%), diabetes (11-31%), and arthritis (10-24%) (4, 17). in this research, 25% of men and 7% of women were treated for alcoholism. it is estimated that 43% of the world's population consumes alcohol (18). in croatia, about 6% of the population suffers from alcohol dependence, which amounts to about 250,000 people (19). spirits (44.8 %) and beer (34.3 %) are most often consumed. alcoholism is twice as common in men than in women, with the highest frequency between the ages of 20 and 24 (18). alcoholics have twice the risk of developing other mental illnesses; these are most often anxiety disorders, affective disorders, personality disorders. psychiatric disorders are thought to precede the development of alcoholism, except for obsessive-compulsive disorder and depression, which occur after alcohol use disorder (20). in addition, there is also a great link with various physical comorbidities. in our study, the most common somatic diseases in patients treated for alcoholism were epilepsy and hypertension. alcohol has a significant effect on the central nervous system, leading to symptoms of mania, depression, and epileptic seizures. chronic alcohol consumption is related to multiple central and peripheral nervous system dysfunctions due to the direct action of alcohol or its derivatives and vitamin deficiencies associated with alcoholism (21). according to data from 2012, alcohol consumption was associated with 5.5% of cancers (7.2% in men and 3.5% in women) and seemedj 2022, vol 6, no 2 somatic illnesses and mental disorders treatment 63 southeastern european medical journal, 2022; 6(2) with 5.8% of deaths in oncological patients (22). the most common cancers associated with alcohol use are cancers of the oral cavity, pharynx, larynx, esophagus, liver, breast, and colon. the carcinogenic potential is linearly dependent on the amount of consumed alcohol (23). alcohol consumption has a dual effect on the cardiovascular system; it has a cardioprotective effect in small doses (one standard drink), but it is harmful in large doses (24). the consumption of two standard drinks is considered to have a protective effect in diabetes mellitus but consuming four standard drinks a day results in negative effects. since most of the alcohol is metabolized in the liver, liver diseases are not uncommon; chronic liver diseases that lead to cirrhosis are the most often (25). primary alcoholic dementia accounts for 10% of all dementias (26). arterial hypertension is the most common somatic disease in this study. it affected 33% of men and 46% of women, a total of 36% of the respondents. hypertension and coronary heart disease cause significant morbidity in patients, reduce the quality of life, and increase treatment costs. psychosocial factors that lead to anxiety disorders play a role in the development of hypertension. increased autonomic excitation via the hypothalamic-pituitary axis leads to an increase in circulating catecholamines, which increases the risk of hypertension and proinflammatory conditions, which in turn leads to the development of coronary heart disease (27). also, hypertension intensifies symptoms of anxiety and the frequency of panic attacks. in one study, a significantly higher incidence of panic attacks was noticed in patients with hypertension (17%) compared to normotensive patients (11%) (28). other anxiety disorders are also common; monitoring cardiac activity or avoiding certain activities often results in reduced quality of life in patients with hypertension (29). diabetes mellitus is the second most common somatic diagnosis in our respondents (22%). we found a higher incidence in women (32%) than in men (18%). 30% of patients with diabetes suffer from a mental disorder. patients with schizophrenia are two to four times more likely to develop diabetes compared to the general population (30). the prevalence rates of depression and anxiety are significantly higher in diabetics; some studies have reported that the risk is increased by as much as 50-100%. the correlation is two-way, depression disorder results from years of uncontrolled or poorly controlled diabetes. depression disorder, on the other hand, activates neurohormonal and neurotransmitter changes that stimulate the hypothalamic-pituitary axis and the adrenal gland, which releases more cortisol and other hormones responsible for insulin resistance (30, 31). the prevalence of diabetes in depressed adult patients is significantly higher in women than in men. a study conducted in saudi arabia reported that 37% of patients with type 1 diabetes mellitus, 37.9% of patients with type 2 diabetes mellitus, and 13.6% of patients with gestational diabetes suffer from depression. another study reported that 46.15% of patients have these comorbidities, of which 36.7% suffer from severe depression. what is more, levels of glycosylated hemoglobin (hba1c) are significantly higher in people with depression compared with those who do not suffer from it (30). epilepsy affected 15% of men and 8% of women, a total of 13% of respondents. it is estimated that the most common mental illness in comorbidity with epilepsy is depression, and its incidence is about 35%. in patients in whom epilepsy is poorly controlled, the incidence is about 50%, while in patients with well-controlled seizures it is about 10 to 20% (31). one study showed that 16.7% of patients with epilepsy have some sort of anxiety disorder; most people had frequent panic attacks (81.2%). older age of patients and later onset of epileptic seizures are associated with a higher incidence of anxiety disorders (32). comorbidities of physical and mental illnesses are a major challenge in medicine. the mutual pathophysiological effect of one disease on another can significantly affect the course and prognosis of the disease. due to the diverse symptoms experienced by people with multimorbidity, diagnoses are often missed, which can lead to major consequences. it is extremely important to improve cooperation in seemedj 2022, vol 6, no 2 somatic illnesses and mental disorders treatment 64 southeastern european medical journal, 2022; 6(2) treatment and the multidisciplinary approach to the patient in order to reduce the number of hospitalizations, emergency interventions, suicides and improve the patients’ quality of life and life expectancy. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. sartorius n. comorbidity of mental and physical disorders: a key problem for medicine in the 21st century. acta psychiatrica scandinavica 2018; 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41(4):365-77. 28. davies sj, ghahramani p, jackson pr, noble tw, hardy pg, hippisley-cox j, yeo ww, ramsay le. association of panic disorder and panic attacks with hypertension. am j med 1999; 107(4):3106. 29. tsartsalis d, dragioti e, kontoangelos k, pitsavos c, sakkas p, papadimitriou gn, stefanadis c, kallikazaros i. the impact of depression and cardiophobia on quality of life in patients with essential hypertension. psychiatriki 2016; 27(3):192203. 30. al-atram aa. a review of the bidirectional relationship between psychiatric disorders and diabetes mellitus. neurosciences journal 2018; 23(2):91-96. 31. kruse j, petrak f, herpertz s, albus c, lange k, kulzer b. diabetes and depression--a life-endangering seemedj 2022, vol 6, no 2 somatic illnesses and mental disorders treatment 66 southeastern european medical journal, 2022; 6(2) interaction. z psychosom med psychother 2006; 52(3):289-309. 32. wiglusz ms, landowski j, cubała wj. prevalence of anxiety disorders in epilepsy. epilepsy behav 2018; 79:1-3. author contribution. acquisition of data: rm, al, dd, tb administrative, technical or logistic support: rm, al, dd, tb analysis and interpretation of data: rm, al, dd, tb conception and design: rm, al, dd, tb critical revision of the article for important intellectual content: rm, al, dd, tb drafting of the article: rm, al, dd, tb final approval of the article: rm, al, dd, tb guarantor of the study: rm, al, dd, tb obtaining funding: rm, al, dd, tb provision of study materials or patients: rm, al, dd, tb statistical expertise: rm, al, dd, tb other: rm, al, dd, tb seemedj 2017, vol 1, no. 2 dietary supplements and sport performance – a comprehensive review 33 southeastern european medical journal, 2017; 1(2) dietary supplements and sport performance – a comprehensive review 1 julij šelb1, helena lenasi2 1 university clinic of respiratory and allergic diseases golnik, golnik, slovenia 2 university of ljubljana, faculty of medicine, institute of physiology, ljubljana, slovenia corresponding author: helena lenasi – helena.lenasi.ml@mf.uni-lj.si received: july 30, 2017; revised version accepted: november 24, 2017; published: november 24. 2017 keywords: dietary supplements, sports, endurance, performance-enhancing substances conclusion. study results showed that agnor count and structure cannot help in determining more clearly the border between parathyroid adenoma and hyperplasia in cytologic smears. (fustar preradovic lj, danic d, kardum-skelin i, sarcevic b, danic hadzibegovic a. agnor counts in differential diagnosis of parathyroid adenoma and hyperplasia in preoperative cytologic smears. seemedj 2017; 1(2); 19-26) abstract aim: comprehensive articles on dietary supplements and their impact on sport performance, which would provide professional and recreational athletes with evidence-based information, are sparse. methods: investigation involved eight different dietary supplements, commonly used among recreational and endurance athletes according to available literature obtained by searching the pubmed database, namely: antioxidants, beta-alanine (b-alanine), branched-chain amino acids (bcaas), caffeine, carbohydrates, creatine, nitric oxide/nitrates, and proteins. their mechanisms of action have been briefly presented, along with their potential beneficial and harmful side effects and safety. results: i.) antioxidants: a sufficient amount of antioxidants is available in a balanced diet ii.) b-alanine: supplementation is likely to be beneficial in high-intensity exercises. iii.) bcaas: no review articles in english were available iv.) caffeine: caffeine supplementation is beneficial in endurance exercises v.) carbohydrates: carbohydrate supplementation is probably beneficial in exercises lasting longer than one hour vi.) creatine: creatine supplementation is effective in high intensity, short-lasting exercise, while it does not seem to have any ergogenic effect in aerobic exercise. vii.) nitric oxide/nitrates: nitrate supplementation has a small but significant performance-enhancing effect, most apparently in situations of insufficient perfusion. viii.) proteins: protein supplementation, in combination with resistance exercise, most likely has beneficial effects on lean body mass and muscle strength. conclusion: most of the analyzed dietary supplements, if used for intended exercise regime, provide a kind of sport performance enhancement. on the other hand, long-term studies about their safety are mostly lacking. šelb j, lenasi h. dietary supplements and sport performance – a comprehensive review. seemedj 2017; 1(2); 33-45) seemedj 2017, vol 1, no. 2 dietary supplements and sport performance – a comprehensive review 34 southeastern european medical journal, 2017; 1(2) abbreviations bcaas – branched-chain amino acids ros reactive oxygen species camp cyclic adenosine monophosphate c-p creatine phosphate no – nitric oxide b-alanine – beta-alanine b-carotene – beta-carotene introduction professional athletes and amateurs alike are in constant search for new means which would enable them to improve their sport results in shorter time. among those means, a prominent place belongs to dietary supplements (1). the use of dietary supplements among athletes varies quite substantially (2–5), and can reach numbers as high as 98% (4), with one of the main reasons for their utilization being the improvement in sport performance (2–5). there are a lot of studies, assessing different dietary supplements and their influence on athletic performance, as well as numerous review articles and meta-analyses evaluating individual supplements or family of supplements (whey proteins, branched-chain amino acids (bcaas), antioxidants…) with respect to sport performance. yet, a systematic review of literature that could give an athlete a more comprehensive overview of the vast and diverse field of dietary supplements is lacking. review articles and meta-analyses are tools used in science to evaluate evidence and also to make scientific conclusions, since they sum up and critically assess the knowledge about a particular topic that is usually dispersed in the form of original articles. the goal of this paper was to gather currently available and evidencebased information in the form of review articles and/or meta-analyses on most currently used sport performance-enhancing dietary supplements and present them to a reader in an understandable manner, as it has been shown that the acquisition of information about dietary supplements usually does not come from evidence-based source of information, but rather from family members, friends and coaches (6). accordingly, we have identified the most commonly used performance-enhancing dietary supplements and studied recently published review articles/meta-analyses about those products, with an aim to write a comprehensive overview of dietary supplements that are used for sport performance enhancement. methods pubmed was searched using the search string “(spor*[title] or athl*[title] or train*[title]) and performan*[title]”, to recognize papers that presented any facts connected to enhancing sport performance (different types of dietary supplements, different exercise procedures …) published in the last five years. analyzing the titles and abstracts of those articles, we identified the most commonly used dietary supplements or groups of dietary supplements associated with enhancing sport performance, and then searched the pubmed library to find review articles/meta-analyses that evaluated each individual dietary supplement or a group of dietary supplements (i.e. string “(carbohy*[title]) and (sport*[title] or athl*[title] or exercis*[title]) and (revie*[title] or analys*[title])” was used to find review articles/meta-analyses focusing on carbohydrate supplements used in sports). if a search string returned many review articles/meta-analyses, just the most recent ones were included in the analysis (and reported in the results section). the reference lists of included articles (articles writing about supplements used to enhance sport performance in general, not inside a specific context [i.e. just endurance sports]) were examined to find additional papers. results eight different dietary supplements or groups of dietary supplements were recognized: i.) antioxidants, ii.) beta-alanine (b-alanine), iii.) seemedj 2017, vol 1, no. 2 dietary supplements and sport performance – a comprehensive review 35 southeastern european medical journal, 2017; 1(2) branched-chain amino acids (bcaas), iv.) caffeine, v.) carbohydrates, vi.) creatine, vii.) nitric oxide (no)/nitrates and viii.) proteins (whey and other proteins). 3.1. antioxidants: the review articles/metaanalyses for the effects of antioxidants were found using the search string “antiox*[title] and (sport*[title] or athl*[title] or exercis*[title]) and (revie*[title] or analys*[title])” on pubmed. the search returned four articles, one of which was about exercise and pregnancy and was therefore excluded from the analysis. out of the three that remained, the most recent one (7) was included in the analysis (it was published in 2015; the other two were written in 1999 and 1993, respectively). another review article, describing vitamin c and its effect on performance, was found while searching through the reference list of the first article and was also included in the analysis. 3.1.1. mechanism(s) of action: during exercise, the production of reactive oxygen species (ros) in the skeletal muscle cells increases (8), and this can have damaging effects as ros can alter cell structure and function, and cause fatigue (9). on the other hand, ros are speculated to be involved in glycogen resynthesis (10) and also in adaptive responses induced by training (11–14). the use of the adequate amounts of antioxidants can therefore, in theory, optimize the balance between pro and anti-ergogenic effects of ros and consequently improve performance. 3.1.2. meta-analysis/revision conclusions: braakhuis and hopkins (2015), who analyzed 71 studies examining the impact of various antioxidants (vitamin e, quercetin, resveratrol, beetroot juice, other food derived polyphenols, spirulina and n-acetylcysteine) on performance, concluded that the only antioxidant exerting beneficial acute effects on performance was nacetylcysteine when injected intravenously, but this route of administration was not recommended (7). the review of braakhuis (2012), which considered 11 articles that examined potential effects of vitamin c on sport performance, found that large doses (more than 1 g/day) of vitamin c appeared to reduce the training-induced adaptations by reducing mitochondrial biogenesis or possibly by altering vascular function (15). they also concluded that small doses of vitamin c (approximately 0.2 g/day), provided by five servings of fruit and vegetables per day, may be sufficient to reduce oxidative stress without reaching the threshold that would impair optimal training adaptations. moreover, a short-term intake (during a period of one to two weeks) of doses greater than 2 g/day seems to be beneficial for athletes during times of increased stress (15). 3.2. b-alanine: the review articles/metaanalyses for the effects of b-alanine were found using the search string “*alanin*[title] and (sport*[title] or athl*[title] or exercis*[title]) and (revie*[title] or analys*[title])” on pubmed. the search retrieved two papers. of the two, the first was a review article written in 2010 (16), and the other one was a meta-analysis conducted in 2012 (17). 3.2.1. mechanism(s) of action: the effects of balanine supplementation can mostly be attributable to an increase in concentration of carnosine (a dipeptide of the amino acids balanine and histidine concentrated in muscle and brain tissue) in the skeletal muscle cells, as concentration of b-alanine is thought to be the rate-limiting step in carnosine synthesis (balanine and l-histidine [carnosine synthase]) (18–20). carnosine is speculated to exert buffering (20), antioxidant (21,22) and calcium (ca2+) regulatory (23,24) effects, that could potentially enhance performance. 3.2.2. meta-analysis/revision conclusions: the meta-analysis was conducted on 15 papers. authors concluded that b-alanine supplementation elicited a significant performance-enhancing effect on high-intensity exercise, particularly when exercise lasted between one and four minutes (17). as b-alanine improved predominantly short-lasting anaerobic exercise performance, the authors argue that those data provide further evidence that supplementation of b-alanine increases intramuscular carnosine, which has ph buffering capacity (17). seemedj 2017, vol 1, no. 2 dietary supplements and sport performance – a comprehensive review 36 southeastern european medical journal, 2017; 1(2) 3.3. bcaas: the review articles/meta-analyses for the effects of bcaas were found using the search string “(bcaa*[title] or branch*[title]) and (sport*[title] or athl*[title] or exercis*[title]) and (revie*[title] or analys*[title])” on pubmed. the search retrieved only one review writing about bcaas and sport performance enhancement (of the nine retrieved by the search, seven were focused on exercise and heart bundle branch blocks, and one was focused on exercise and dissection of the celiac trunk and its branches), as it was written in spanish, we only assume the proposed mechanisms of bcaas actions. 3.3.1. mechanism(s) of action: bcaas are thought to improve performance by diminishing the exercise-induced increase in serotonin, which is thought to be at least partly responsible for the central feeling of fatigue during exercise (25). the exercise-induced increase in serotonin is thought to be induced by an increase of plasma free tryptophan. the concentration of free tryptophan rises during exercise because of increased levels of non-esterified fatty acids, the levels of which are also higher during exercise, and which compete with tryptophan for the same binding sites on albumin (26). the transport of tryptophan across the blood-brain barrier is considered to be the rate-limiting step in the synthesis of serotonin. bcaas compete with tryptophan in that transport (27). 3.3.2. meta-analysis/revision conclusions: the search string on pubmed returned only one article, which was in spanish, so we have no results to report in this section. 3.4. caffeine: the review articles/meta analyses for the effects of caffeine were found using a search string: “(caffei*[title]) and (sport*[title] or athl*[title] or exercis*[title]) and (revie*[title] or analysi*[title])” on pubmed. the search returned seven articles. according to the titles and abstracts of those articles, none was appropriate (some of them were dealing with caffeine and sport performance in a specific setting [endurance performance, high intensity performance, …] others were about pharmacological aspects of caffeine, while another one dated as far back as to the year 1994), so we included a review (28) article in the “similar articles” section in the pubmed. 3.4.1. mechanism(s) of action: caffeine exerts various effects on many organ systems. it is an inhibitor of phosphodiesterase (increasing the intracellular cyclic adenosine monophosphate [camp]) and also an antagonist of adenosine receptors (its psychoactive effect) (29). its actions on sport performance are thought to be mediated through its effects on the cardiovascular system (an increase in heart rate and blood pressure ), the pulmonary system, the endocrine system and the central nervous system (28). 3.4.2. meta-analysis/revision conclusions: authors of the review (28) concluded that caffeine is beneficial in endurance exercise, as it has been shown to increase work output and the time to exhaustion. they also stated that it should be ingested one hour before prolonged endurance events and that, if an athlete decides to stop consuming caffeine before competition with an aim to increase its ergogenic effects during competition (since a person can get habituated), one should reduce caffeine consumption at least one week before the competition, to be completely free from its withdrawal effects. furthermore, to avoid potential negative symptoms, the dose should gradually be reduced over three to four days, instead of quitting abruptly. resuming caffeine on the day of competition can again provide the desired ergogenic effects, similarly as it would for a nonuser (28). they have also concluded that because caffeine increases the plasma concentration of lactate and hence decreases ph, it may be contraindicated in athletes engaging in sprint events which last between 30 seconds and three minutes (28). 3.5. carbohydrates: the review articles/meta analyses for the effects of carbohydrates were found using a search string “(carbohy*[title]) and (sport*[title] or athl*[title] or exercis*[title]) and (revie*[title] or analysi*[title])” on pubmed. the search returned nine articles; yet, only one was appropriate for inclusion in the analysis (30), since others were seemedj 2017, vol 1, no. 2 dietary supplements and sport performance – a comprehensive review 37 southeastern european medical journal, 2017; 1(2) either about carbohydrates taken in combination with proteins, or about the effects of carbohydrates on lipid metabolism and about different ways of use of carbohydrates (mouth rinse). 3.5.1. mechanism(s) of action: there are two proposed mechanisms when trying to explain how carbohydrate supplementation during exercise improves performance (30): i.) mental cognitive stimulation of the central nervous system by carbohydrate exposure during exercises of shorter duration (less than one hour the glycogen stores are not a limiting factor) (31–33) and ii.) a direct exogenous carbohydrate contribution to carbohydrate oxidation during muscle glycogen limiting exercises (duration longer than two hours) (34–36). 3.5.2. meta-analysis/revision conclusions: the revision included 61 different performance studies performed on 679 subjects. eighty-two percent of the studies showed performance benefit, while 18 % showed no performance improvement compared to placebo (30). based on the revision of the studies, authors recommend that for exercises of shorter duration (less than one hour) and high intensity, small amounts of liquid carbohydrate solutions provide performance benefit through stimulation of the pleasure and reward center in the brain (30). by increasing the duration of exercise (duration of one to two hours), moderate amounts of carbohydrate supplementation (30-60 g/h) consumed frequently throughout the exercise appear to maximize the performance advantage of carbohydrate supplementation (liquid carbohydrate sources are recommended (30)). with the duration of exercise extending beyond two hours (with concurrently reduced intensity), a greater variety of carbohydrate foods and fluids can be included to meet the high hourly carbohydrate intake recommendations of 40110 g/h (30). 3.6. creatine: the review articles/meta analyses for the effects of creatine were found by using a search string: “(creati*[title]) and (sport*[title] or athl*[title] or exercis*[title]) and (revie*[title] or analysi*[title])” on pubmed. the search string retrieved seven articles, four of which were appropriate (they were about sport performance in general with regard to creatine use), with the most recent one dating to 2003 (37). 3.6.1. mechanism of action: creatine supplementation is thought to increase intramuscular storage of creatine phosphate (cp), which is responsible for anaerobic atp resynthesis during high-intensity, short-duration exercises (38,39). based on the available data, positive correlations between muscle creatine uptake and exercise performance (40) were shown. 3.6.2. meta-analysis/revision conclusions: based on the available research data, authors concluded that creatine supplementation could increase the skeletal muscle c-p content, which may improve performance involving short periods of extreme exertion , especially during repeated bouts of exercise (37). on the other hand, creatine supplementation did not appear to increase the maximal isometric strength, the rate of maximal force production, or the aerobic exercise performance (37). 3.7. nitrates: the review articles/meta-analyses for the effects of nitrates were found using a search string: “(nitr*[title]) and (sport*[title] or athl*[title] or exercis*[title]) and (revie*[title] or analysi*[title])”. the search retrieved five articles: only two (41,42) of them were appropriate (dealing with nitrate supplementation and exercise performance), while others were mainly focused on other issues, such as the effects of exercise and nitrates on vascular endothelial function, the effects of nitrates on exercise-induced bronchospasm, etc.), so the most recent one (42) was analyzed. 3.7.1. mechanism of action: it is believed that inorganic nitrate can be converted to its antecedents (nitrite and no), especially in the presence of hypoxia and acidosis (41). no is thought to exert multiple effects in the body, including its effects on neurotransmission (43), on vascular tone control (44), mitochondrial respiration (45) and skeletal muscle contraction (46), thus its influence on exercise is complex. seemedj 2017, vol 1, no. 2 dietary supplements and sport performance – a comprehensive review 38 southeastern european medical journal, 2017; 1(2) 3.7.2. meta-analysis/revision conclusions: the authors state that nitrate supplementation has no or little effect on maximal oxygen consumption (vo2max) but it can lower the oxygen deficit of submaximal exercise (that is, improve skeletal muscle efficiency or economy), at least in subjects who are not highly trained (42). this may lead to a small increase in the sustainable power output for a given metabolic rate. they also conclude that at least in situations where some fraction of the working muscle mass may be relatively under-perfused (in hypoxia, during high-intensity exercise in subjects with low aerobic fitness when their muscles are relatively under-perfused with respect to the heart) (42), nitrate supplementation can speed up the oxygen kinetics and thus have an ergogenic effect. the optimally efficient nitrate dose is likely to vary between subjects but is hypothesized to be generally more pronounced in highly trained individuals (42). 3.8. proteins (whey): the review articles/meta analyses for the effects of proteins were retrieved using the search string: “(protei*[title]) and (sport*[title] or athl*[title] or exercis*[title]) and (revie*[title] or analysi*[title])” on pubmed. the search returned 27 articles, three of which were appropriate for analysis (dealing with protein supplementation and sport performance), and since the most recent one (47) was funded by a commercially orientated consortium, the second most recent one was chosen for analysis (48). 3.8.1. mechanism(s) of action: protein balance in the human body depends on protein synthesis and protein degradation. when the synthesis is higher than the breakdown, one will have a positive protein balance, otherwise the protein balance will be negative (49). amino acid intake (50,51) and resistance exercise training (52–56) have separately been shown to have a positive effect on protein synthesis. moreover, their combined effect (i.e. ingesting proteins after resistance training) was shown to be greater than just adding two separate effects together (57–59). 3.8.2. meta-analysis/revision conclusions: the meta-analysis consisted of 22 randomized controlled trials that, in the aggregate, included 680 subjects (48). authors concluded that dietary protein supplementation represented an effective dietary strategy to augment the adaptive response of skeletal muscle to prolonged resistance-type exercise training in healthy younger (younger than 50) and older (older than 50) adults. furthermore, in younger adults, protein supplementation during prolonged (more than six weeks) resistancetype exercise training significantly augmented the gains in fat-free mass, type i and ii muscle fiber cross-sectional area, and one-repetition maximum leg press strength compared to resistance-type exercise training without a dietary protein-based cointervention (48). the findings in the group of younger adults were evident, despite the fact that, before the intervention, all groups were already consuming a more than adequate dietary protein intake of 1.2 g/kg body weight/day (48). from a practical point of view, it is worth noting that subjects were supplemented with an average of 50 ± 32 g proteins / day (in excess of their normal diet) and, in most cases, the protein supplements were ingested before or immediately after each exercise session. discussion in the era of highly competitive sports, more and more individuals are using performanceenhancing nutritional supplements to enhance not only their sport performance but also their visual appearance and self-confidence. consumers of those supplements are overwhelmed by the manufacturers’ claims of increased strength, weight loss, and improved body definition, but information about the efficacy is rarely acquired or presented in an evidence-based manner (6). there is a large body of information in the form of original articles and/or review articles and meta-analyses about a particular nutritional supplement, but a more comprehensive overview of the vast and diverse field of dietary supplements is lacking. keeping that in mind, a review article was written, identifying the common sport performance-enhancing dietary seemedj 2017, vol 1, no. 2 dietary supplements and sport performance – a comprehensive review 39 southeastern european medical journal, 2017; 1(2) supplements, finding the relevant and recent review articles/meta-analyses about those supplements, and summarizing their findings and conclusions. eight dietary supplements/groups of dietary supplements were identified and focused on: antioxidants, b-alanine, bcaas, caffeine, carbohydrates, creatine, no/nitrates and proteins (whey and other proteins). regarding antioxidants, of all the tested antioxidants (vitamin e, quercetin, resveratrol, beetroot juice, other food-derived polyphenols, spirulina and n-acetylcysteine), the authors found strong evidence for performanceenhancing effect only for n-acetylcysteine (but with the very impractical administration route, namely i.v.) (7). the authors also speculated that antioxidants could reduce the impairment of performance during tournaments or repeated efforts, presumably by reducing the severity of inflammation (7). similar speculations were hypothesized in a review about the influence of vitamin c on performance (15), where the authors concluded that the amount obtained in five servings of fruit and vegetables would be sufficient to reduce oxidative stress, and that higher doses might be beneficial only during periods of intensified stress (15). accordingly, based on the conclusions of those two reviews, the amount of antioxidants we get in a balanced diet is probably sufficient, even for athletes, and thus, the supplementation of antioxidants should be considered only in periods of increased stress (tournaments, world series). the safety of long term antioxidant use should also be taken into account, when considering antioxidant supplementation for enhancing sport performance, since a double-blinded, placebo-controlled study about the use of vitamin a and beta-carotene (b-carotene) and their effect on lung cancer, the beta-carotene and retinol efficacy trial, showed, on 18314 participants, that there was a significantly higher relative risk for lung cancer and lung cancer associated mortality among the patients who were receiving a combination of b-carotene and vitamin a, compared to placebo (60). the results of this study were concordant with the results of the randomized, double-blinded, placebocontrolled trial conducted on 29133 finnish smokers, which examined the effects of alphatocopherol and/or b-carotene, and found an eight percent higher overall mortality among patients receiving b-carotene (compared to the ones who did not receive it) (61). the results of a recently published cochrane meta-analysis (approximately 300,000 participants) which compared the effects of antioxidant supplements (beta-carotene, vitamin a, vitamin c, vitamin e, and selenium) versus placebo or no intervention, were also worrying as they found that b-carotene and vitamin e seemed to increase mortality; a similar possibility was suggested for larger doses of vitamin a (62). as for b-alanine supplementation, the authors of a meta-analysis conducted in 2012 (17) concluded that b-alanine supplementation had an ergogenic effect on high intensity exercises lasting between one and four minutes, possibly contributable to ph buffering capacity of intramuscular carnosine, the concentration of which rises with supplementation. the authors of a 2010 review (16) also noted similar observations, concluding that b-alanine carries potentially beneficial effects in high-intensity exercise including anaerobic sprints and resistance training. b-alanine supplementation is thus likely to be beneficial in high-intensity exercises. a systematic review from 2014 (63) found similar results regarding performance, indicating that b-alanine may increase power output and working capacity, decrease the feeling of fatigue and exhaustion, and have positive effects on body composition and carnosine content. in addition, this study concentrated on side effects of b-alanine supplementation, and found that they were mostly mild, consisting of paraesthesia and/or infrequent mild transient symptoms such as tingling in hands and fingers. the study also stressed the need for more studies dealing with long term effects of b-alanine supplementation. the ergogenic effect of caffeine on endurance performance was demonstrated by multiple meta-analyses/review articles which also concluded that in habituated caffeine consumers, the abstinence from caffeine at least seven days before competition would give the seemedj 2017, vol 1, no. 2 dietary supplements and sport performance – a comprehensive review 40 southeastern european medical journal, 2017; 1(2) greatest chance of optimizing its ergogenic effect (28,64). however, the effect of caffeine on high-intensity performance is not well established, since caffeine increases plasma lactate concentration and hence decreases ph (28). on the other hand, a systematic review (65) of the effect of acute caffeine ingestion on shortterm high-intensity exercise performance showed that 11 (of 17) studies revealed significant improvements in team sports exercise and power-based sports after caffeine ingestion, while further six (of 11) studies showed significant benefits for resistance training. based on the results of the above studies, caffeine supplementation for endurance exercises seems to be beneficial, but when it comes to the effects of caffeine on high-intensity exercise, further investigation is required. long-term ingestion of moderate doses of coffee (three to four coups [less than 400 mg/day]), is suggested to have mostly beneficial effects and has been shown to be inversely associated with the risk for various diseases (66). epidemiological data support the view that habitual coffee consumption lowers the risks of parkinson’s and alzheimer’s disease, has a favorable effect on liver function, a possible role in weight loss (66), and also decreases the risk for developing certain types of cancer (endometrial, prostatic, colorectal, liver) (67,68). on the other hand, coffee intake has been associated with bone loss and adverse effects in pregnancy (66). nevertheless, the growing body of evidence from epidemiological studies supports the notion that moderate coffee consumption exerts mostly beneficial effects on health and reduces mortality (67). unfortunately, the association does not show causality, so large double-blinded, placebo-controlled studies are needed to clarify the effects of coffee consumption on health per se. moreover, the effects of coffee consumption are not attributable solely to caffeine (since coffee has more than 1,000 different compounds (66)), while the supplements that are used to increase sport performance use mostly caffeine as an active compound, and lack other compounds found in a cup of coffee. consequently, to determine the safety of caffeine-containing sport performance-enhancing supplements, additional studies on those supplements should be more thoroughly evaluated before giving adequate evidence-based statements about the safety of those supplements. several systematic reviews (30,31) confirmed the efficacy of carbohydrate mouth rinse on exercise performance with activities lasting less than one hour, potentially attributable to activation of pleasure and reward center in the brain (30). as for ingestion of carbohydrates, a systematic review (69), which included only studies mimicking real life situations (subjects exercising in the postprandial state) and excluding studies that followed rigorous criteria like keeping athletes in a fasted state, concluded that carbohydrate supplementation during exercise bouts of less than 70 minutes is unlikely to be beneficial (69). on the other hand, a beneficial effect of carbohydrate supplementation during exercise lasting more than 70 minutes was confirmed in a systematic review that included all types of studies (those mimicking real life situations and also studies following rigorous inclusion criteria) (30) and also in the review (69) that included just studies mimicking real life situations. the most probable beneficial effect was attributed to the replacement of exhausted glycogen storage (30, 69). taken together, carbohydrate supplementation is probably beneficial in exercises of longer duration (more than one hour), while in short-lasting exercise (less than one hour), more research is required to determine the efficacy in ‘real-life situations mimicking’ scenarios. as for creatine, several review articles/metaanalyses (37,70) showed that it was effective in increasing performance in high-intensity, shortlasting (less than 30 seconds) exercises, and also that the effect of creatine diminished with increasing duration of exercise (70). it seems that creatine does not have any ergogenic effect on aerobic exercise (37). short-term administration of creatine supplementation seems to be safe, since studies have not found clinically significant deviations from normal values in the renal, hepatic, cardiac, gastrointestinal or muscle function (71,72). in fact, most reports on its seemedj 2017, vol 1, no. 2 dietary supplements and sport performance – a comprehensive review 41 southeastern european medical journal, 2017; 1(2) potential side effects, such as muscle cramping, gastrointestinal symptoms, changes in renal and hepatic laboratory values, remain anecdotal (72). studies which would evaluate the safety of longterm creatine supplementation are lacking. a few studies that examined the long-term effect of creatine supplementation on kidney function (73), or on various blood and urinary markers of health (metabolic markers, skeletal muscle and liver enzymes, electrolytes, lipid profile, hematological markers, and lymphocytes) (74), showed no differences between the measured outcomes in the creatine-taking group compared to the control group. additional studies on larger number of participants are needed to give firm evidence-based conclusions regarding long-term safety of creatine supplements. regarding nitrate supplementation, the analyzed review articles/meta-analyses (41,42) have concluded that nitrate supplementation had a small but significant (41) performanceenhancing effect. the effect was most apparent in situations where skeletal muscles were insufficiently perfused, such as during hypoxia, or high-intensity exercise in subjects exhibiting low vo2max, when the contracting muscle is in a disadvantageous position relative to the heart and is relatively under-perfused (42). a recent study has shown that short-term nitrate supplementation (up to 28 days) was safe, and all hematological safety markers remained in a normal range (75), but long-term studies regarding safety are needed. the effects of protein supplementation (50 ± 32 g/day [above the usual diet]) were shown to be beneficial in augmenting the gains of fat-free muscle mass, type i and ii muscle fiber crosssectional area, and muscle strength, especially in combination with resistance training of longer duration (more than six weeks) (48). on the other hand, another review (76) showed that for untrained individuals, consuming supplemental proteins most likely had no impact on lean mass and muscle strength during the initial weeks of resistance training. however, as the duration, frequency, and volume of resistance training increase, protein supplementation may promote muscle hypertrophy and enhance gains in muscle strength in both untrained and trained individuals (76). additionally, a third metaanalysis (47) showed similar beneficial effects of whey protein consumption on body composition parameters, which were most pronounced with resistance training exercise. we may conclude that protein supplementation, in combination with resistance exercise of longer duration (more than six weeks), most likely has beneficial effects on body composition (lean body mass) and possibly also on muscle strength. mounting evidence suggests that protein intake in excess of two to three times the recommended daily allowance may have harmful effects on the homeostasis of calcium and, possibly, bone mass, particularly for proteins from primarily animal sources and in situations where potential loss of bone calcium has not been minimized by additionally supplementing other dietary elements such as potassium or bicarbonate. additional potential harmful effects of high-protein intake on kidneys, the cardiovascular system, and carcinogenesis have been suggested but available data remain inconclusive, and further research is necessary (77). conclusion most of the revised dietary supplements, if used for the intended exercise regime, provide some sort of sport performance enhancement. sport performance enhancement can be a clear-cut one, or it can be observed only in strict laboratory conditions, it may involve unusual routes of administration or may only be successful in a subset of people. nevertheless, at least in professional sport, where milliseconds decide between success and failure, the competitive edge that dietary supplements can bring, can give an athlete an advantage to succeed. on the other hand, the safety of longterm supplementation, being another and even more important aspect, should also be taken into account when considering whether or not performance-enhancing supplements should be encouraged and consumed. unfortunately, seemedj 2017, vol 1, no. 2 dietary supplements and sport performance – a comprehensive review 42 southeastern european medical journal, 2017; 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4(1) original article influence of nodal yield in individual neck dissection levels on survival of patients with oral and oropharyngeal cancer 1 ana kvolik 1, 2, josip butković 1, 2, vedran zubčić 1, 2, zvonimir popović 2, 3, dinko leović* 2, 4, 5 1 department of maxillofacial and oral surgery, university hospital osijek, croatia 2 faculty of medicine, josip juraj strossmayer university of osijek, osijek, croatia 3 department of neurology, university hospital osijek, croatia 4 department of ent and head and neck surgery, university hospital centre zagreb, croatia 5 faculty of dental medicine and health, josip juraj strossmayer university of osijek, osijek, croatia *corresponding author: dinko leović, dinko.leovic@gmail.com received: oct 15, 2019; revised version accepted: jan 13, 2020; published: apr 27, 2020 keywords: oral cancer, oropharyngeal cancer, neck dissections, lymph node excisions, disease-free survival, kaplan-meier estimate abstract introduction: nodal yield (ny), or the number of collected and analysed lymph nodes in neck dissection, is one of the variables that could supplement the existing tnm classification in order to better stratify patients and their needs for further treatment. the purpose of this paper was to investigate the importance of ny in individual neck dissection levels and its relation to survival. materials and methods: a retrospective analysis of medical records of 133 patients regarding primary tumour excision and neck dissection from 2002 to 2013. seventy-nine patients had a neck dissection divided by levels at the time of surgery and 54 patients had an en bloc resection. results: in the group of all patients, there was no correlation between ny and survival. in the group of patients who underwent a selective neck dissection, a ny above the median was an indicator of a better disease-specific survival (5-year dss < median ny 70.6%, > median ny 95.2%, p = 0.037 logrank test). the ny of specimens separated by level was significantly higher than the ny of specimens analysed en bloc (median 33 vs 16; p < 0.001, median test). in the group of specimens separated by level, the ny in levels i-ii was not associated with survival, but a high ny in levels iii-iv in selective neck dissections was an indicator of an improved overall survival (p = 0.05), disease-specific survival (p = 0.022) and disease-free survival (p = 0.05). conclusion: high ny in patients with specimens separated by levels could be caused by a more precise pathohistological analysis of a smaller sample. a high ny in levels iii-iv can be an indicator of a well-performed selective neck dissection and sufficiently treated regional disease and therefore lead to better survival rates. (kvolik a, butković j, zubčić v, popović z, leović* d. influence of nodal yield in individual neck dissection levels on survival of patients with oral and oropharyngeal cancer, comorbidity and chronic therapy. seemedj 2020; 4(1); 14-24) seemedj 2020, vol 4, no. 1 nodal yield and survival with oral and oropharyngeal cancer 15 southeastern european medical journal, 2020; 4(1) introduction oral and oropharyngeal squamous cell carcinoma is one of the most prevalent malignancies of the head and neck1,2. its treatment has changed significantly in the last decades, after the introduction of novel chemo/chemoradiotherapeutic protocols, as well as a result of more sparing surgical procedures. it is well-known that oral and oropharyngeal cancer first metastasises in the regional lymph nodes of the neck (figure 1). figure 1. levels of neck dissection as metastases in the lower neck levels are quite rare, selective neck procedures have been developed. a sparing approach could provide a good postoperative recovery and minimal function impairment3, but could also result in understaging of the disease due to the presence of an undetected metastasis or micrometastasis4. the question pertaining to the adequacy of the extent of neck dissection, especially when it comes to selective dissection, remains. nodal yield (ny), or the total number of excised and pathohistologically examined lymph nodes, is a means of quantifying the extent of neck dissection, but it is still unclear whether it is also an independent criterion for survival. the value of nodal yield in clinical practice has been discussed in recent studies5,6,7, but the results so far have been inconclusive. the aim of this study was to elucidate the connection between nodal yield and survival and the possibility of stratification of high-risk patients based on nodal yield. materials and methods a retrospective study of the influence of nodal yield on the outcome of patients treated surgically due to oral or oropharyngeal cancer was conducted. all patients were treated at the same department of the clinical hospital centre from 1 january 2002 to the end of 2013 and their tumours were classified according to the 2002 tnm staging rules8. the study was approved by the institutional ethics committee (number r2:22512-6/2015 clinical hospital centre osijek, ethics committee). patients’ medical records and the hospital’s electronic database were analysed. general inclusion criteria were the following: pathohistological diagnosis of oral or oropharyngeal squamous cell carcinoma, surgical treatment including a radical or selective neck dissection and follow-up of at least two years or until death. from 229 patients diagnosed with oral or oropharyngeal carcinoma in this period, 133 were included in the study. there were 17 patients whose neck specimens had descriptive pathohistological terms such as “several”, “a few” or “conglomerates” of lymph nodes and as such, they were unfit for statistical analysis. thirty-one patients were treated only with transoral excision without neck dissection due to the early stage of the disease and were not included in the study. the remaining 48 patients were lost to follow-up or refused a proposed therapy. nodal yield (ny) was defined as the total number of lymph nodes analysed in a neck specimen. if a patient underwent a bilateral dissection, ny was calculated as the sum of lymph nodes on both sides divided by two. other variables noted and used in the statistical analysis were clinical and pathohistological n status, t status, locoregional or distant recurrence and type of seemedj 2020, vol 4, no. 1 nodal yield and survival with oral and oropharyngeal cancer 16 southeastern european medical journal, 2020; 4(1) dissection. finally, the study included only the patients with regard to whom the absolute number of lymph nodes was indicated regardless of their quantity. inclusion criteria were fulfilled by 133 patients on whom 149 neck dissections have been performed. among them, 118 patients received postoperative radio and/or chemoradiotherapy, while four patients received preoperative radioand/or chemoradiotherapy. due to a poor general condition, medical contraindications, a prolonged postoperative recovery or early stage of the disease, 10 patients did not receive any adjuvant radio-/chemoradiotherapy. statistical analysis all statistical analyses were performed for the all-patients group and subgroups divided by surgical treatment, cn status and pn status. the above-mentioned groups were dichotomised by the median (due to the irregularity of spread of ny values) and cutoff point of 18 lymph nodes according to the recent report of ebrahimi et al., who found a minimum ny of 18 to be a marker of a well-performed elective selective neck dissection and of a patient’s outcome5,9. a separate statistical analysis was additionally made only for the patients (n = 79) whose neck specimens had been divided by levels at the time of surgery. unfortunately, there was no consistency in the manner of division between various surgeons, e.g. some surgeons divided the upper neck levels (i and ii) and lower neck levels (iii and iv) as one sample, whilst others separated each level. in order to uniform our data, we presented the results regarding nodal yield in the regions of the upper neck and lower neck, while level v, present only in radical dissections, was analysed separately. we are aware that running multiple statistical analyses on a small data sample increases the probability of a chance finding, which is one of the major limitations of this study. however, it is our opinion that dividing patients into smaller groups was necessary in order to get the data that accurately portrays each specific group of patients. the statistical analysis was performed using the spss 22.0 statistical software (spss inc., chicago, il, usa). two-year and five-year overall survival (os), disease-free survival (dfs) and disease-specific survival (dss) were calculated using the kaplan-meier survival method (log-rank, breslow and tarone-ware tests). os was calculated as the time from the treatment to the last follow-up or death, dfs as the time from the treatment to the recurrence of carcinoma, either locoregional or as a metastasis, and dss as the time from treatment to death due to oral or oropharyngeal scc. a univariate analysis using the pearson’s chisquare test was made for os, dfs and dss. for the data with expected cell frequencies ≤ 5, the fisher’s exact test was performed. medians were compared with the median test. level of significance was defined as p < 0.05 and all statistical tests that were used in the calculations were two-sided. results from january 2002 to december 2013, 133 patients who were treated for oral or oropharyngeal cancer at the clinical hospital centre osijek met the inclusion criteria, of whom 122 (91.7%) were male and 11 (8.3%) were female. the mean age was 57.8 ± 8.6 years. the data regarding localisation, stage and treatment of the tumours is shown in table 1 and figure 2.. table 1. demographical data regarding the study population seemedj 2020, vol 4, no. 1 nodal yield and survival with oral and oropharyngeal cancer 17 southeastern european medical journal, 2020; 4(1) figure 2. localisation of the primary tumour. a) tongue b) tongue base c) mouth floor d) mandibular gingiva e) retromolar f) tonsils g) uvula h) oropharyngeal wall neck dissection and nodal yield from 133 patients, 16 had a bilateral neck dissection, yielding a total of 149 dissections in this study. median nodal yield for the entire study population was 25, with the lowest nodal yield being 4 and the highest being 75. thirtyeight patients underwent a selective neck dissection, while 95 had a radical neck dissection. median nodal yield was 25 for radical and 29 for selective neck dissection. there were 54 patients with the cn0 stage and 79 patients with cn+ neck specimens. in the cn0 group, the median nodal yield was 22 lymph nodes, and in the cn+ group, the median ny was 28 lymph nodes, which could have been a result of more sparing neck dissections being performed on patients with the clinically negative neck. we also analysed the patients according to the pathohistological n status. there were 32 patients whose cn and pn status did not match. forty-six patients had the pn status with a median ny of 29, while 87 patients had the pn+ status with a median ny of 24. nodal yield for each group can be found in table 2.. n % tumour site oral 89 66.9 oropharyngeal 44 33.1 localisation tongue 45 33.8 sublingual 25 18.8 tonsils 20 15.0 tongue base 18 13.5 mandibular gingiva 5 3.8 uvula 7 5.3 retromolar 8 6.0 oropharyngeal wall 5 3.8 t t1 8 6.0 t2 49 36.8 t3 42 31.6 t4 34 25.6 n n0 54 40.6 n1 40 30.1 n2a 10 7.5 n2b, n2c 25 18.8 n3 4 3.0 neck dissection selective 38 28.6 radical 95 71.4 chemoradiotherapy postoperative 118 88.7 preoperative 5 3.8 no treatment 10 7.5 seemedj 2020, vol 4, no. 1 nodal yield and survival with oral and oropharyngeal cancer 18 southeastern european medical journal, 2020; 4(1) table 2. nodal yield in each group or dissection level group (n) median ny overall (133) 25 selective neck dissection (38) 29 radical neck dissection (95) 25 cn0 (54) 22 cn+ (79) 28 pn0 (46) 29 pn+ (87) 24 specimens en bloc (54) 16 specimens separated by level (79) 33 level i-ii (79) 14 level iii-iv (79) 9 level v (57) 8 nodal yield and separation of neck specimens by levels in 79 patients, the neck specimen was separated by levels at the time of surgery by two experienced surgeons. in this group, the median nodal yield was 33, while the group with en bloc resections had a median nodal yield of 16. the difference was statistically significant (p < 0.001, median test). see table 2. survival analysis median survival for the entire group of patients was 33 (17-67) months after the surgery. in the first two years after the surgery, overall survival rate was 61.1% for patients with a ny below the median and 63.8% for patients above the median (p = 0.298; log-rank test). disease-specific survival (the kaplan-meier curve in figure 3) showed similar results (p = 0,103; log-rank test). figure 3. kaplan-meier curves of diseasespecific survival in the entire study population (n = 133); 2-year survival for ny > median group (n = 69) was 70.3%, and for ny < median group (n = 64) 67.7% (p = 0.103 log-rank test, p = 0.334 breslow test, p = 0.206 tarone-ware test) seemedj 2020, vol 4, no. 1 nodal yield and survival with oral and oropharyngeal cancer 19 southeastern european medical journal, 2020; 4(1) for the purposes of survival analysis, we dichotomised all groups according to the nodal yield above and below the median and above and below 18 lymph nodes. there was no difference in survival below and above the cutoff point of 18 lymph nodes in either selective or radical neck dissection. in the group of selective neck dissections (n = 38), the patients with a ny above the median had a statistically better disease-specific survival as opposed to those with a ny below the median (5-year dss < median ny 70.6%, > median ny 95.2%, p = 0.037 log-rank test), as seen in figure 4. figure 4. kaplan-meier curves of disease specific survival for selective neck dissections; 2-year survival for ny > median group (n = 21) was 95.2%, and for ny < median group (n = 17) 70.6% (p = 0.037, log-rank test, p = 0.035 breslow test, p = 0.036 tarone-ware test) however, in the group of radical neck dissections (n = 95), the kaplan-meier estimates did not confirm a statistical significance of nodal yield as an independent predictor of survival (5year dss < median ny 66.6%, > median ny 69.3%, p = 0.459). we found no statistical difference of survival of patients with a higher and lower ny by dividing the patients into the cnand cn+ group in any survival study, but a statistical significance was found when we analysed the pngroup. in patients with pathohistologically negative neck specimens, dss was statistically higher in those patients whose ny was above the median (5year dss < median ny 38.4%, > median ny 67.4%, p = 0.045), while dfs was leaning towards a statistical significance (p = 0.057). ny by neck level and survival median nodal yield was 14 lymph nodes in the upper neck (levels i and ii) and 9 lymph nodes in the lower neck (levels iii and iv). level v, present only in radical dissections, yielded an average of 8 lymph nodes. we reviewed the upper neck ny separately for cn-, cn+, pnand pn+ groups, selective dissection, radical dissection and the entire group, but no statistical correlation between survival and ny was found, only a tendency (p = 0.072) for a slightly better diseasespecific survival of those with a ny above the median in the cngroup. however, ny had interesting properties pertaining to the lower neck. in the group of selective dissections (n = 22), a ny above the median was related with a better os (p = 0.05), dss (p = 0.022), and dfs (p = 0.05). with regard to the radical neck dissection group (n = 57), findings were quite different. interestingly, we found worse outcomes in patients with a ny above the median as compared to a ny below the median (os p = 0.009, dss p = 0.014, dfs p = 0.007). kaplan-meier plots can be seen in figure 5. seemedj 2020, vol 4, no. 1 nodal yield and survival with oral and oropharyngeal cancer 20 southeastern european medical journal, 2020; 4(1) figure 5. kaplan-meier curves of disease-specific survival for ny in lower neck region (3 and 4) in a) selective neck dissections; 2-year survival for > median group (n = 12) was 100%, and for the < median group (n = 10) 30.9% (p = 0.022 log-rank test, p = 0.044 breslow test, p = 0.029 tarone-ware test), and b) radical neck dissections; 2-year survival for > median group (n = 17) 38.0% and for < median group (n = 40) 56.8% (p = 0.014, log-rank test, p = 0.006 breslow test, p = 0.007 tarone-ware test) with regard to level v, the results varied, with the lowest nodal yield being 0 and the highest being 27, which was not statistically significant for survival in any group of patients. discussion even though the gold standard treatment for oral and oropharyngeal carcinoma includes a resection of the primary tumour followed by a neck dissection, the consensus pertaining to the width of the neck dissection has not been reached since its introduction in 1906. squamous cell carcinoma usually metastasises in the upper neck levels (i, ii, iii), so the procedures have been shifting to a more sparing and selective neck dissection in order to preserve the function and cause minimal impairment to the patient. since the introduction of adjuvant chemoor chemoradiotherapy, surgical treatment options have become even more selective. however, neck dissection is not only vital for treatment, but also for correct staging. consequently, an over-selective neck dissection could lead to understaging and locoregional recurrence due to missed lymph nodes in the lower levels or due to micrometastases. the problem of the occult neck disease has been widely studied by woolgar and cho et al., who claim that comprehensive neck dissection is an important factor for finding micrometastases in lymph nodes of the neck that cannot be discovered by a routine pathohistological analysis4,10. this raises the issue of re-evaluating the margins of a wellperformed neck dissection and determining how many levels are enough to ensure the best survival, the least regional recurrence rate and minimal impairment. recently, nodal yield has been proposed as a supplement to the standardised tnm classification, as well as a means of quantification of a performed dissection. even though neck dissection is a standardised procedure, nodal yield in each neck dissection varies dramatically. this could be caused by its dependence on the three following criteria: width of the dissection, the level of pathologists’ seemedj 2020, vol 4, no. 1 nodal yield and survival with oral and oropharyngeal cancer 21 southeastern european medical journal, 2020; 4(1) scrutiny and individual differences in lymphatic tissue. patel et al. reported a nodal yield of 2-104 (a mean of 39 ± 23) nodes11, ali et al. 7-140 (a mean of 42) nodes12 and ebrahimi reported a mean ny of 25.59, 276 and 305 in different studies. the median ny of 25 found in this study does not vary significantly compared to other studies. nodal yield depends even on the type of surgical technique. thus, lörincz et al. 13 proved that a standardised horizontal dissection of the cervical fascia yields more lymph nodes than a caudal-to-cranial dissection. it is noteworthy to add that all our neck dissections have been performed in a standardised horizontal manner and included level i, both for oral and oropharyngeal cancer. several studies have tried to analyse ny and its impact on survival, but the results have so far been indecisive. ebrahimi et al. 9 showed that a nodal yield above 18 is an independent prognostic factor for patients undergoing a selective neck dissection for cn0 oral squamous cell carcinoma. as this study population included patients undergoing both a radical and a selective neck dissection, in all n stages of the disease, we divided the patients into multiple groups in order to homogenise the sample. we found no statistically significant difference in survival of any group of patients with a cut-off point of 18, which could be explained by a higher t and n status at the beginning of the treatment, but also as an inter-institutional difference of overall ny between studies. lemieux studied cnpatients, divided ny into quartiles and found an improved outcome in two higher quartiles (ny > 22)14. our study used the median of each group as a cut-off point and showed similar results as lemieux et al.14 in the pngroup with a ny above the median having a better disease specific survival as compared to those with a ny below the median. these findings could support the theory that a higher ny increases the likelihood of finding neck metastases and allows for adequate clearance of occult metastases. nodal yield is also dependent on the manner of presenting the specimen to the pathologist. in a traditional radical neck dissection, en bloc specimen with non-lymphatic tissue such as the sternocleidomastoid muscle and jugular vein would be presented to the pathologist who was charged with dissecting the specimen by level and analysing it. this procedure was quite imprecise as the tissue of the sample is prone to shrinkage and the identification of level borders was often inaccurate, but this practice became even more imprecise with the introduction of selective neck dissections15,16. in selective neck dissection, samples did not have any anatomical structure that could be used for orientation. a correct analysis of each level is an important factor that provides information about the course of future treatment17. dividing specimens by levels at the time of surgery is, therefore, the key to better and accurate staging; it reduces the manipulation of the tumour and tumour spillage. smaller, more manageable samples could allow the pathologist to find and analyse more lymph nodes and to detect micrometastases if they are present15,16. this was confirmed by our data, with a much lower median ny of 19 in en bloc specimens, as compared with a median ny of 35 in specimens divided by levels. however, not all studies have come to the same conclusion; kerawala17 et al. found no significant difference between en bloc and divided specimens, while marres et al.18 noticed an increase in ny when specimens were examined by a pathological technician as compared to an examination by a pathologist. to the best of our knowledge, no one has studied the importance of nodal yield in relation with survival on each level. kerawala et al. 17 reported a median ny in each level as follows: i3, ii-9, iii-7, iv-5 and v-9, while norling et al.19 studied nodal yield in cadavers as compared to other reports from literature. both studies showed a notoriously wide range of nodal yield in each level (varying from 0 up to > 20). our data, therefore, does not vary greatly from other reports. as described in the materials and methods section, we presented nodal yield separately for the upper neck (levels i and ii) and the lower neck (levels iii and iv). the data regarding nodal yield in the lower neck levels was particularly perplexing, especially regarding radical neck dissections having a lower ny than selective neck dissections. this difference might be caused by an imprecise separation, which led seemedj 2020, vol 4, no. 1 nodal yield and survival with oral and oropharyngeal cancer 22 southeastern european medical journal, 2020; 4(1) to lymph nodes from levels iii and iv to end in level v specimens. it remains unclear why radical neck dissection overall had a slightly lower ny than selective dissection, but this could be due to differences in the number of samples (94 radical neck dissections as compared to 38 selective neck dissections). skip metastases, or metastases present only in the lower neck levels without involvement of levels i and ii, remain a controversial topic and one of the arguments for a radical surgical treatment. while feng et al.20 report a rate of 1.1% for skip metastasis in level iii and 3.2% in level iv, they concluded that supraomohyoid dissection does provide adequate care for patients with oral scc. khafif et al.21 also argued that dissection of level iv is necessary only when there is intraoperative suspicion of metastatic involvement in levels ii and iii. dias et al.22 reported only 2% of skip metastasis, but 24.1% of occult neck metastases. it is, therefore, very important to correctly weigh the need to perform a selective or radical neck dissection. in this study, nodal yield in the upper neck showed no statistical significance pertaining to survival. this could be caused by easy surgical access and relatively uniform clearance of this level. a high nodal yield in the lower neck, however, proved to be an important predictor of outcome for node-negative and node-positive neck specimens treated by selective neck dissections. these findings are in consistence with other studies in which the positivity of the lower neck regions was found to be a predictor of a worse outcome23 and could mean that by dissecting level iv, skip metastases, as well as occult metastases, have been cleared. the group of patients undergoing radical neck dissections showed quite an opposite result, where patients with a higher ny had a statistically worse outcome. patients undergoing a radical neck dissection belonged to a group of a higher t and n stage. therefore, it is logical that higher ny dissections would reveal more positive lymph nodes, which negatively affects the outcome. it is rather difficult to compare selective and radical neck dissections because of various other factors that affect a patient’s prognosis, such as a more advanced disease, higher risk of perioperative complications and locoregional or distant failure, which are not directly linked with nodal yield. it is also important to evaluate the role of all healthcare providers who participated in the treatment of our patients, such as pathologists, whose impact on ny could not be assessed in detail in this study. however, the number of patients in whom neck specimens were divided into levels and who could partake in the statistical analysis was too small to make any definitive conclusions, but the conclusions that could be made raise some interesting questions and deserve further investigations in a larger cohort study. conclusion our data showed improved outcomes of welltreated, high ny patients as compared with those with a lower ny. this could indicate the need to review the strategies of surgical approach to the neck metastasis, which has recently shifted towards a more selective approach. a better outcome in patients with a higher ny in the lower neck, even in the pn group, could mean a higher probability of clearance of occult metastases, which have been linked to a worse outcome10. even so, our data on the importance of ny in the lower neck must be treated with caution because of a small sample, but it could indicate a trend that should be studied in more detail in a larger study. acknowledgement. none. disclosure. this research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. this study was conceived as the final diploma work of the first author (ak) and was presented at graduation. preliminary results were presented at the 12th congress of croatian society of maxillofacial, plastic and reconstructive surgery of the head and neck; 2-4 june 2016, osijek, croatia seemedj 2020, vol 4, no. 1 nodal yield and survival with oral and oropharyngeal cancer 23 southeastern european medical journal, 2020; 4(1) references 1. more y, d’cruz ak. oral cancer: review of current management strategies. natl med j india. 2013;26(3):152-158. http://www.ncbi.nlm.nih.gov/pubmed/244 76162. 2. cohan dm, popat s, kaplan se, rigual n, loree t, hicks wl. oropharyngeal cancer: current understanding and management. curr opin otolaryngol head neck surg. 2009;17(2):88-94. doi:10.1097/moo.0b013e32832984c0. 3. liang l, zhang t, kong q, liang j, liao g. a meta-analysis on selective versus comprehensive neck dissection in oral squamous cell carcinoma patients with clinically node-positive neck. oral oncol. 2016;51(12):1076-1081. doi:10.1016/j.oraloncology.2015.10.005. 4. woolgar ja. micrometastasis in oral/oropharyngeal squamous cell carcinoma: incidence, histopathological features and clinical implications. br j oral maxillofac surg. 2016;37(3):181-186. doi:10.1054/bjom.1999.0037. 5. ebrahimi a, clark jr, amit m, et al. minimum nodal yield in oral squamous cell carcinoma: defining the standard of care in a multicenter international pooled validation study. ann surg oncol. 2014;21(9):3049-3055. doi:10.1245/s10434-014-3702-x. 6. ebrahimi a, clark jr, zhang wj, et al. lymph node ratio as an independent prognostic factor in oral squamous cell carcinoma. head neck. 2011;33(9):1245-1251. doi:10.1002/hed.21600. 7. gil z, carlson dl, boyle jo, et al. lymph node density is a significant predictor of outcome in patients with oral cancer. cancer. 2009;115(24):5700-5710. doi:10.1002/cncr.24631. 8. deschler d, day t. tnm staging of head and neck cancer and neck dissection classification. alexandria am acad head neck surg. 2008. http://webmail.entnet.org/educationandr esearch/upload/neckdissectionpart1.pdf. 9. ebrahimi a, zhang wj, gao k, clark jr. nodal yield and survival in oral squamous cancer. cancer. 2011;117(13):2917-2925. doi:10.1002/cncr.25834. 10. cho j-h, lee y-s, sun d-i, et al. prognostic impact of lymph node micrometastasis in oral and oropharyngeal squamous cell carcinomas. head neck. 2016;38(s1):e1777e1782. doi:10.1002/hed.24314. 11. patel sg, amit m, yen tc, et al. lymph node density in oral cavity cancer: results of the international consortium for outcomes research. br j cancer. 2013;109(8):20872095. doi:10.1038/bjc.2013.570. 12. amar a, rappaport a, curioni oa, dedivitis ra, cernea cr, brandao lg. the density of metastatic lymph node as prognostic factor in squamous cell carcinoma of the tongue and floor of the mouth.br j otorhinolaringol.2012;78(3):86-90. 13. lörincz bb, langwieder f, möckelmann n, sehner s, knecht r. the impact of surgical technique on neck dissection nodal yield: making a difference. eur arch oto-rhinolaryngology. 2016;273(5):1261-1267. doi:10.1007/s00405-015-3601-1. 14. lemieux a, kedarisetty s, raju s, orosco r, coffey c. lymph node yield as a predictor of survival in pathologically node negative oral cavity carcinoma. otolaryngol -head neck surg. 2016;154 (3 ):465-472. doi:10.1177/0194599815622409 . 15. jose j, coatesworth ap, maclennan k. cervical metastases in upper aerodigestive tract squamous cell carcinoma: histopathologic analysis and reporting. head neck. 2003;25(3):194-197. doi:10.1002/hed.10194. 16. coatesworth ap, maclennan k. squamous cell carcinoma of the upper aerodigestive tract: the prevalence of microscopic extracapsular spread and soft tissue deposits in the clinically n0 neck. head neck. 2002;24(3):258-261. doi:10.1002/hed.10020. 17. kerawala cj, bisase b, hopper a. is total nodal yield in neck dissections influenced by seemedj 2020, vol 4, no. 1 nodal yield and survival with oral and oropharyngeal cancer 24 southeastern european medical journal, 2020; 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7(1) invited review anti-inflammatory and antioxidant response in covid-19 infection: nrf2/ho-1 pathway 1 lana maričić 1,2*, damir mihić 1,2, nikolina šego 3 1 department of internal medicine, university hospital centre osijek, osijek, croatia 2 faculty of medicine, josip juraj strossmayer university, osijek, croatia 3 health center of osijek-baranja county, osijek, croatia *corresponding author: lana maričić, lmaricic@mefos.hr received: mar 7, 2023; revised version accepted: apr 6, 2023; published: apr 30, 2023 keywords: covid-19, sars-cov-2, heme oxygenase-1, nrf2 factor abstract sars-cov-2 virus infection starts with the internalization of the viral particle into the host cells, mainly the upper respiratory system epithelial cells which have the highest expression of the ace2 receptor which is essential for the internalization process. the pathophysiology of severe forms of covid-19 disease results not only from direct, cytopathic viral effect but also from immune response dysregulation of the host resulting in hyperinflammatory state and oxidative stress. the nuclear factor erythroid 2-related factor 2 (nrf2) ability to protect cells and induce a rapid anti-inflammatory and antioxidant response primarily depends on its constitutive cellular expression, which can be affected by numerous endogenous and exogenous factors. the binding of nrf2 to cellular receptors leads to the transcription of a large number of genes encoding various antioxidant enzymes and other cytoprotective molecules, including heme oxygenase-1(ho-1). activation of ho-1 results in antioxidant, anti-inflammatory and anti-apoptotic effects. based on previous studies, the nrf2/ho-1 pathway provides protection against oxidative stress and inflammatory and immune response which is significant in covid-19 infection, which is characterized by a strong hyperinflammatory response. this narrative review aims to describe the role of the hyperinflammatory response in the development of covid-19 infection, with a focus on the nrf2/ho-1 pathway. (maričić l*, mihić d, šego n. anti-inflammatory and antioxidant response in covid-19 infection: nrf2/ho-1 pathway. seemedj 2023; 7(1); 1-12) seemedj 2023, vol 7, no 1 nrf2/ho-1 pathway in covid-19 infection 2 southeastern european medical journal, 2023; 7(1) introduction coronaviruses belong to the rna viruses group with the largest genome among the rest of the rna viruses (27–37 kb). virus particles have a characteristic membrane with spikey structured proteins on the outer surface, which is where the name coronavirus (lat. corona, crown) comes from. three groups of coronaviruses differ genotypically, of which viruses from group iii are found only in birds, while viruses from groups i and ii are also found in mammals. coronaviruses mainly cause respiratory system diseases in humans, and at least four variants continuously circulate within the population, especially among young children. two variants were identified in the mid-1960s (hcov-oc43 and hcov-229e), and the other two in the early 2000s (hcov-nl-63 and hcov-hku1). the first coronavirus linked with a more serious illness was identified at the end of 2002. due to its tendency to cause severe acute respiratory syndrome (sars), it was named sars-cov. it’s believed to be of wild animals origin, most likely bats, and was transmitted to humans via infected bats that were prepared and sold as food in china [1]. the sars-cov epidemic has affected 29 countries, 8.096 cases were registered, and 774 deaths were confirmed, according to which the estimated mortality was 9.6%. ten years later, in april 2012., another coronavirus associated with severe forms of respiratory infections emerged, and due to its middle eastern origin, this strain has been named mers-cov (middle east respiratory syndrome). concurrently with the sporadic isolation of mers-cov, in december 2019., a new coronavirus named sars-cov-2 was isolated in the city of wuhan, china. and rapidly spread, not only in china but also in other continents, therefore becoming the first coronavirus that caused a global pandemic[2,3]. disease background despite numerous research, the sars-cov-2 origin remains unclear. the most common theory of this virus origin is associated with bats, which are natural reservoirs of the virus, especially considering the high homology of the genome of viruses isolated from bats with the human sars-cov-2 virus, which amounts to 96.2%. initially, infections of humans by the sars-cov-2 virus appeared as a classic zoonosis since most of the first confirmed coronavirus cases had direct contact with animals at the market in wuhan. the first patients were believed to be infected by consuming infected animals that were prepared as food or sold alive, with bats being the most frequently involved [4]. the rapid spread and appearance of the disease among healthcare workers and people who did not have direct contact with infected animals indicated humanto-human transmission. since it’s mainly a respiratory disease and viruses are isolated from the epithelial cells of the respiratory tract, it can be assumed that the main transmission route of the virus between people is droplet transmission, i. e. through aerosols created when speaking, sneezing or coughing [6,5]. although sars-cov-2 has been detected in urine, stool, blood/serum, saliva, and tears, there is no strong evidence that it can be transmitted in such a manner. structure and pathogenesis of sars-cov-2 infection the sars-cov-2 genome size is 29.9 kb, and two-thirds of the genome encodes proteins necessary for virus replication. one-third encodes structural proteins: protein e (envelope protein), protein s (spike protein), protein m (membrane protein), and protein n (nucleocapsid protein). proteins s, m, and e form the virus particle envelope, and protein n, which binds the rna molecule to itself, is found inside the virus particle [7,8]. the sars-cov-2 genome is highly variable, resulting from errors that occur during rna molecule replication, whereby they accumulate and create new mutations, resulting in the emergence of new viral subvariants or strains. to date, more than 20 strains of this virus have been described. as some mutations cause pathogenicity or virulence modifications (either positive or negative ones), the world health organization identifies five variants of concern (vocs), given their public health impact: alpha (b.1.1.7) which was originally isolated in the uk in late december 2020., beta (b.1.351), which was seemedj 2023, vol 7, no 1 nrf2/ho-1 pathway in covid-19 infection 3 southeastern european medical journal, 2023; 7(1) first reported in south africa in december 2020., gamma (p.1), which was first reported in early january 2021. in brazil, delta (b.1.617.2), first reported in december 2020. in india and omicron (b.1.1.529), first reported in south africa in november 2021. [9]. both pathogenicity and virulence of the sars-cov-2 result from its ability of cellular internalization, i.e., the ability of the virus to enter cells. the entry of the sarscov-2 virus into the cells is mediated by the s protein of the virus particle and the ace2 receptor (angiotensin-converting enzyme 2) of the host cells. ace2 receptor was identified in 2003. and the sars-cov-2 and other coronaviruses (hcov-229e, hcov-oc43 and hcov-hku1) are uptaken via aforementioned receptor. protein s contains two subunits: the s1 subunit enables viral particle binding to the ace2 receptor via the receptor-binding domain (rbd), while the s2 subunit enables the fusion of the viral particle and the host cell membranes [10]. in addition to the ace2 receptor, integrins and cd174-sp are also mentioned as potential receptors for the internalization of sars-cov-2 into host cells. there is no strong experimental evidence for these assumptions [11,12]. since ace2 has a wide biodistribution in the organism, including the respiratory system, digestive system (small and large intestine), myocardium, kidneys, thyroid gland, liver, brain, and olfactory neuroepithelium, sars-cov-2 has a broad tissue and cell tropism, resulting in wide clinical presentation and a clinically diverse manifestation [13]. despite the broad distribution of these receptors, the respiratory system is predominantly and most severely affected by this viral infection. this can be explained by the fact that the respiratory system epithelial cells have the largest expression of ace2 receptors, as well as the fact that the main transmission route of the disease is droplet, and the respiratory system mucous membrane is the initial site for the virus entrance into the body. epithelial cells of nasal mucosa and bronchial epithelial cells are the site of the largest ace2 receptor expression, while in the alveoli the ace2 receptor is found only in type ii pneumocytes [14]. upregulated expression of the ace2 receptor in the respiratory system epithelial cells is affected by proinflammatory cytokines that are produced during infection, especially il-1β and type i and iii interferons, which can accelerate its spread and replication [15]. clinical characteristics covid-19 infection the respiratory system is the main organ system affected by this disease, but due to broad sarscov-2 virus tropism, other clinical manifestations are possible as the result of other tissues and organs involvement. it’s a highly contagious illness that has a wide clinical spectrum, that varies from asymptomatic to mild, severe, and critical (29). clinically, covid19 infection is most often presented as a common cold or a flu-like illness, which in certain individuals can progress to more severe forms, leading to pneumonia and acute respiratory distress syndrome, and also to manifestations that are caused by other tissues and organs involvement [16]. acute respiratory distress syndrome (ards) represents the most severe form of the respiratory system involvement by the sars-cov-2 virus and is accompanied by a high rate of severe complications such as sepsis, septic shock, and multiorgan failure, all of which increase the mortality rate. ards is characterized by newonset severe respiratory insufficiency and bilateral pulmonary infiltrates, which occurs as a result of still insufficiently elaborated pathophysiological mechanisms that include the direct viral cytopathic effect, immune response dysregulation (hyperinflammatory state or cytokine storm) as well as increased oxidative stress. the final result is extensive lung parenchyma damage and consequently severely impaired oxygen and carbon dioxide exchange (hypoxemia, hypercapnia) [17]. as previously discussed, sars-cov-2 predominantly affects the respiratory system, but other organs and organ systems can also be affected, either as a single organ disease, but most common with the respiratory system involvement. the most common extrapulmonary covid-19 manifestations involve gastrointestinal and hepatobiliary, hematological, cardiac, renal, and neurological systems [16]. seemedj 2023, vol 7, no 1 nrf2/ho-1 pathway in covid-19 infection 4 southeastern european medical journal, 2023; 7(1) immune response in covid-19 infection sars-cov-2 virus infection starts by the internalization of the viral particle into the host cells, mainly the upper respiratory system epithelial cells which have the highest expression of the ace2 receptor which is essential for the internalization process. the viral entry into the cells triggers an immune response whose role is to neutralize the virus and bring the infection under the control. today, it’s considered that the pathophysiology of severe forms of covid-19 infection results not only from direct, cytopathic viral effect but also from immune response dysregulation of the host resulting in hyperinflammatory state and oxidative stress [18]. by viral particle entrance and the viral rna release in the host cell cytoplasm begins a cellular response, in addition to the process of virus replication. the cellular response aims to activate the immune response as a protective mechanism of the host against the virus [19]. in this process prr receptors (pattern recognition receptors) are essential. these are found in epithelial cells cytoplasm and have role of recognizing pamp molecules (pathogen-associated molecular patterns) [20]. these prr receptors recognize long chains of viral rna that are formed as intermediates during replication, causing their activation and triggering signaling pathways that include interferon regulatory factor (irf) and nuclear factor-κb (nfκb) [21]. irf promotes the transcription of interferons i and iii, while nf-κb promotes the transcription of pro-inflammatory cytokines and chemokines [22]. interferons, proinflammatory cytokines, and chemokines are responsible for chemotaxis and mutual interaction of innate immunity cells: neutrophils, monocytes that differentiate into macrophages, dendritic cells and nk cells. they form the first line of defense of the organism against the sars-cov-2 virus. in addition to innate immunity cells, t and b lymphocytes also participate in the immune response to the sars-cov-2 virus. after phagocytosis of infected epithelial cells, antigen presenting cells (apc), among which respiratory dendritic cells dominate move to regional lymph nodes and present there the viral antigen, which is expressed on their surface trough molecules of the mhc complex (major histocompatibility complex), to the naïve circulating t lymphocytes. viral antigens are recognized by t-cell receptors (tcr) of naïve t lymphocytes, and as a result, their interaction leads to their sensitization, whereby they are activated, proliferate, and migrate to the infection localization. at the infection localization activated cytotoxic t lymphocytes (cd8+) produce antiviral cytokines (inf-γ, tnf-α, il-2) that inhibit viral replication, chemokines (cxcl9, 10 and 11) that enhance the chemotaxis of other effector t lymphocytes and cytotoxic molecules (perforin, granzyme b) that kill infected cells, thus trying to eliminate the virus. in addition to classic cd4+ and cd8+ lymphocytes, the so-called unconventional lymphocytes t (ut) that accumulate in the mucous membranes, especially of the respiratory system, are also a part of immune reaction against the sars-cov-2 virus. they represent a heterogeneous t lymphocytes group that can recognize non-peptide viral antigens and are not limited to the presentation of antigens via the mhc system like the classic lymphocytes [23]. the humoral immune response is the most effective and long-lasting immune mechanism in terms of host defense against viruses, including the sars-cov-2 virus. it’s based on the interaction of naïve b lymphocytes with antigen and cd4+ t lymphocytes, the ultimate consequence of which is maturation into plasma cells and the production of specific antibodies [24]. in the host’s battle with the sars-cov-2 virus antibodies targeted at the rbd domain of the s protein are the most effective because they most successfully block the binding of the virus particle to the ace2 receptor on the cell surface [25]. hyperinflammatory immune response and cytokine storm in covid-19 infection immune system dysregulation in response to the presence of the sars-cov-2 virus, and not only the cytopathic effect of the virus, is responsible for development of more severe clinical forms of the covid-19 infection [18]. hyperinflammatory response is a condition seemedj 2023, vol 7, no 1 nrf2/ho-1 pathway in covid-19 infection 5 southeastern european medical journal, 2023; 7(1) resulting from uncontrolled activation of the immune system, which is primarily manifested by increased production and release of proinflammatory cytokines, which is known in clinical practice as a cytokine storm or cytokine release syndrome. the current focus in the development of hyperinflammatory syndrome pertains to dysregulation of the host’s innate immunity response cells in the presence of the sars-cov-2 virus [26]. the basis of this theory is pyroptosis, the programmed cell death of infected cells, which is activated during viral rna replication. this is why pyroptosis is also known as an inflammatory form of programmed cell death. during pyroptosis, various molecules that can function as stimulators of innate immunity cells are released [27,28]. among these is il-1β, whose high levels are detected in the serum and bronchoalveolar lavage of patients suffering from severe forms of covid19 disease [29]. by disruption of infected cells during pyroptosis parts of viral rna that act as pamp molecules (pathogen-associated molecular patterns) and other viral proteins are released. these are recognized by previously described prr receptors of the surrounding cells and trough the il-1r signaling pathway stimulate transcription and production of proinflammatory cytokines such as il-6, il-8, il10, tnf-α, inf-γ, mcp, mip and others. they lead to chemotaxis of macrophages and other effector cells of innate immunity as well as t lymphocytes which further enhance the immune response, cause local damage of cells and tissues and additionally produce cytokines resulting in constant amplification and stimulation of the immune response. finally, produced cytokines also enter the circulation and can cause damage in distant tissues and organs, which explains the occurrence of multiorgan failure in critical patients with covid19 [30,31]. the hyperinflammatory state or cytokine storm is considered one of the main factors in the development of the most severe forms of covid-19 infection, which is acute respiratory distress syndrome (ards) and associated multiorgan failure [32]. multiorgan failure is promoted by activation of endothelial cells mediated by tnf-α and il-1β, which brings to increased expression of selectin p, fibrinogen and von willebrand factor, as well as induced tissue factor release, which initiates the coagulation cascade and the formation of clots, especially at microcirculatory level [33]. oxidative stress in covid-19 infection the production of reactive oxygen species and oxidative stress is one type of the host response to the presence of viruses in its cells and is part of the overall defense against infection, due to active participation of reactive oxygen species in elimination of pathogens. also, several reactive oxygen species are involved in intercellular and intracellular signaling pathways of the immune system cells [34]. there are several assumed mechanisms of increased reactive oxygen species concentration in covid-19 infection. the general mechanism is based on the reninangiotensin-aldosterone system, that is, the interaction of angiotensin ii and nadph oxidase. angiotensin ii is known as a stimulator of nadph oxidase, which generates reactive oxygen species and oxidative stress. under physiological conditions, ace2 catalyzes the conversion of angiotensin ii to angiotensin, which doesn't affect nadph oxidase, but moreover, has an antioxidative effect. during the sars-cov-2 virus infection, the availability of “free” ace2 decreases due to its binding to the virus and/or entry into cells. the result is an increased concentration of angiotensin ii and nadph oxidase stimulation, which causes an increased production of reactive oxygen species and oxidative stress [35,36]. therefore, the inflammatory reaction intensity and the degree of innate immune response dysregulation (excessive activation of monocyte-macrophage system cells) are proportional to oxidative stress level, and reactive oxygen species together with proinflammatory cytokines lead to the celltissue damage present in patients suffering from severe forms of covid-19. nrf2/ho-1 signaling pathway in covid-19 infection the relation between the hyperinflammatory state and oxidative stress represents a great challenge in patients suffering from covid-19 seemedj 2023, vol 7, no 1 nrf2/ho-1 pathway in covid-19 infection 6 southeastern european medical journal, 2023; 7(1) infection, especially in terms of discovering molecular pathways that could potentially be therapeutic targets and whose modulation could reduce the inflammatory response and oxidative stress, essential elements in the development of severe forms of covid-19 disease (figure 1). figure 1. nrf2/ho-1 signaling pathway in covid-19 infection the transcriptional factor nrf2 (nuclear factor erythroid 2-related factor 2, nrf2) is one of the most frequently researched molecules in terms of the cellular anti-inflammatory and antioxidant response in viral infections. the ability of nrf2 to protect cells and induce a rapid antiinflammatory and antioxidant response primarily depends on its constitutive cellular expression, which can be affected by numerous endogenous and exogenous factors [37]. the basic binding site of nrf2 is the promoter region of dna, are region (antioxidant response element), responsible for the transcription of genes whose products are part of the antioxidant response [38]. the binding of nrf2 to the are region leads to the transcription of a large number of genes encoding various antioxidant enzymes and other cytoprotective molecules. these are enzymes that participate in the metabolism of iron and heme (heme oxygenase-1), enzymes that participate in the direct neutralization of reactive oxygen species (nadph quinone reductase, superoxide dismutase, catalase, glutathione peroxidase, glutathione transferase), as well as enzymes that participate in the synthesis and regeneration of glutathione and nadph (glutamate cysteine ligase, glutathione reductase, glucose-6phosphate dehydrogenase) [39]. in addition to the previously discussed effect of nrf2 by binding to the are region, there are also findings of its direct anti-inflammatory effect, independent of gene expression. kobayashi et al. described in their research that nrf2 can reduce the expression of pro-inflammatory cytokines (il-1, il-6) in activated macrophages by directly inhibiting their transcription in the cell nucleus [40]. heme oxygenase-1 (ho-1) is commonly researched enzyme that is produced as a result of the interaction of nrf2 and the are region. it is involved in the catabolism of prooxidant heme, which results in numerous effects, including antioxidant, anti-inflammatory and anti-apoptotic effects. ho-1 catalyzes the reaction that breaks down heme and produces iron (fe2+), which is stored in the cell in the form of ferritin, carbon monoxide (co) and biliverdin. biliverdin is quickly converted by biliverdin reductase into bilirubin, which has higher electrophilicity than biliverdin and a stronger antioxidant effect. in addition to heme degradation products, ho-1 also has direct beneficial effects that may have certain implications for the development of the covid19 infection. the antiviral effect of ho-1 is based on several mechanisms. the basic mechanism involves an increase in the interferon i level, which has been proven to be suppressed in cells infected with the sars-cov-2 virus. interferon i, in addition to interfering with viral rna replication, also enhances the cytolytic effect of innate immunity cells against cells infected with the sars-cov-2 virus [41]. studies on animal models also indicate a direct anti-inflammatory effect of ho-1 mediated by a reduction in the production of pro-inflammatory cytokines [42]. considering the antioxidant, anti-inflammatory and antiviral effects of nrf2, its reduced concentration can result in increased oxidative stress, enhanced immune response, as well as faster and uncontrolled virus replication [40,43]. seemedj 2023, vol 7, no 1 nrf2/ho-1 pathway in covid-19 infection 7 southeastern european medical journal, 2023; 7(1) clinical implications of nrf2/ho-1 pathway in covid-19 infection, possible therapeutic approach research shows that dysregulation of the immune system and increased expression of oxidative stress are essential in the development of the most severe forms of the covid-19 infection [26,44], therefore reduced nrf2 values result in a worse clinical course and severe clinical forms of the disease. the key question research has addressed is the mechanisms of nrf2 activation. a possible explanation is that the nrf2 expression is mainly affected by endogenous or exogenous stimulus, and not by its spontaneous transcription because its expression is inhibited in basal conditions, which is why its activation is also one of the cellular defense mechanisms [45]. viremia acts as a direct activator of nrf2, and it could be expected that as a result, the serum concentrations of nrf2 are higher in viral infections [46]. the activation of nrf2 results in antiviral activity, some authors assume that its lower values in viral infections, especially in severe ones, may also be the result of a direct viral effect on its reduced expression in order to insure a smoother replication [38]. the presence of complications such as bacteriemia and sepsis can inhibit nrf2 activation [47-49]. this is in line with research conducted on patients with covid-19 infection [50-52] in which patients with the most severe form of the disease have lower nrf2 values. when analyzing other exogenous activators of nrf2, 1,25-dihydroxy vitamin d should be pointed out in covid-19 infection. in line with numerous studies lower values of 1,25-dihydroxy vitamin d are associated with a worse outcome, a more severe clinical picture, considering that a lower value of the vitamin is correlated with reduced nrf2 activation. as proven in numerous studies, in patients with covid-19 infection, nrf2 is affected by the oxygen therapy, which leads to increased oxidative stress and inflammatory response and has an activating effect on nrf2 [53-56]. oxygen administration is required in severe forms of the disease, which results in additional oxidative stress that increases nrf2 expression. however, patients with the most severe forms of the disease have lower nrf2 values. one of the explanations is that the application of mechanical ventilation i.e. longer exposure to fio2 results in decreasing in antioxidant and anti-inflammatory effects, and the period and expression of nrf2 [52,57]. although for now there is not much clinical research, these have indicated the correlation of serum ho-1 level with worse clinical outcomes and clinical findings [58]. a large amount of evidence, primarily based on animal models, support the idea that nrf2 and ho-1 can provide protection against oxidative stress as well as inflammatory and immune responses [59]. many studies indicate the importance of nrf2 and its equivalents in embryonic development, stress signaling and aging [60]. moreover, nrf2dependent genes, such as ho-1 provide a cytoprotective effect and play a crucial role in the development of oxidative and aging-related disorders. a more detailed analysis of micrornas involvement in nrf2/ho-1 regulation could provide new ideas for the treatment of many diseases (malignant, autoimmune). the involvement of nrf2 in reductive stress in an animal model of protein aggregation cardiomyopathy indicates the contribution of this transcriptional regulator of antioxidant genes in other pathological situations related to unbalanced redox homeostasis. due to animal models, our knowledge of the nrf2/ho-1 pathway in controlling physiology and disease progression advanced significantly, but additional clinical research concerning individual pathological conditions is needed. in this review, we systematically presented the immune response as part of the covid-19 infection. we focused on the development of anti-inflammatory and antioxidant responses, with an emphasis on the nrf2/ho-1 pathway. it should be emphasized that clinical studies have been described, the connection with the severity of the disease, using mechanical ventilation. the above is intended to encourage future research to consider a therapeutic approach through nrf2 activation. seemedj 2023, vol 7, no 1 nrf2/ho-1 pathway in covid-19 infection 8 southeastern european medical journal, 2023; 7(1) conclusion one of the important features of covid-19 infection is hyperinflammatory response and due to it, the role of the nrf2/ho-1 pathway is extremely important. previous research proposed many mechanisms that positively or negatively affect the activation of nrf2 and ho1, as well as their correlation with inflammatory response markers. all further studies concern the factors that affect its activation, including the exogenous factors, as well as the consequences of the adverse effects of the patient’s comorbidities on the activation of the nrf2/ho1 pathway. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. ksiazek tg, erdman d, goldsmith cs, zaki sr, peret t, emery s, tong s, urbani c, comer ja, lim w, rollin pe, dowell sf, ling ae, humphrey cd, shieh wj, guarner j, paddock cd, rota p, fields b, derisi j, yang jy, cox n, hughes jm, leduc jw, bellini wj, anderson lj; sars working group. a novel coronavirus associated with severe acute respiratory syndrome. n engl j med. 2003;348(20):1953-66. doi: 10.1056/nejmoa030781. 2. azhar ei, hui dsc, memish za, drosten c, zumla a. the middle east respiratory syndrome (mers). infect dis clin north am. 2019 dec;33(4):891-905. doi: 10.1016/j.idc.2019.08.001. 3. zhou p, yang xl, wang xg, hu b, zhang l, zhang w, si hr, zhu y, li b, huang cl, chen hd, chen j, luo y, guo h, jiang rd, liu mq, chen y, shen xr, wang x, zheng xs, zhao k, chen qj, deng f, liu ll, yan b, zhan fx, wang yy, xiao gf, shi zl. a pneumonia outbreak associated with a new coronavirus of probable bat origin. nature. 2020;579(7798):270-273. doi: 10.1038/s41586020-2012-7. 4. li h, tao z, yang y, deng z, liu b, ma z, zhang y, shi g, lam tty, wu jt, gao gf, cowling bj, yang b, leung gm, feng z. early transmission dynamics in wuhan, china, of novel coronavirusinfected pneumonia. n engl j med. 2020;382(13):1199-1207. doi: 10.1056/nejmoa2001316. 5. ralp r, lew j, zeng t. 2019-ncov (wuhan virus), a novel coronavirus: human-to-human transmission, travel-related cases, and vaccine readiness. j infect dev ctries. 2020;14:3–17. doi: 10.3855/jidc.12425. 6. wong scy, kwong rt, wu tc, chan jwm, chu my, lee sy, wong hy, lung dc. risk of nosocomial transmission of coronavirus disease 2019: an experience in a general ward setting in hong kong. j hosp infect. 2020;105:119-27. doi: 10.1016/j.jhin.2020.03.036. 7. lu r, zhao x, li j, niu p, yang b, wu h, wang w, song h, huang b, zhu n, bi y, ma x, zhan f, wang l, hu t, zhou h, hu z, zhou w, zhao l, chen j, meng y, wang j, lin y, yuan j, xie z, ma j, liu wj, wang d, xu w, holmes ec, gao gf, wu g, chen w, shi w, tan w. genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. lancet. 2020;395(10224):565574. doi: 10.1016/s0140-6736(20)30251-8. 8. wu a, peng y, huang b, ding x, wang x, niu p, meng j, zhu z, zhang z, wang j, sheng j, quan l, xia z, tan w, cheng g, jiang t. genome composition and divergence of the novel coronavirus (2019-ncov) originating in china. cell host microbe. 2020;27(3):325-328. doi: 10.1016/j.chom.2020.02.001. 9. aleem a, akbar samad ab, slenker ak. emerging variants of sars-cov-2 and novel therapeutics against coronavirus (covid-19). in: statpearls [internet]. treasure island (fl): statpearls publishing; 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revised version accepted: dec 6, 2019; published: dec 16, 2019 keywords: angiography, optical coherence tomography, vasculogenesis, angiogenesis, fovea centralis abstract optical coherence tomography angiography (oct-a) is a new non-invasive technology for imaging of retinal and choroidal vasculature of the macular area with resolution comparable to histological sections. oct-a does not require usage of intravenous dye, contrary to fluorescein angiography, the current gold standard for imaging of retinal vessels, and indocyanine-green angiography, which is important for imaging of choroidal vessels. with the advancements in optical coherence tomography (oct) scanning speeds and creation of powerful algorithms for improvement of image quality in recent years, oct-a imaging of macular vasculature, superficial, deep and avascular retinal complex, as well as choriocapillaris and deep choroid has become available in everyday clinical practice. this review covers aspects important for understanding choroidal and retinal blood supply, as well as the development, mechanisms and clinical application of oct and oct-a technology. (bajtl d, bjeloš m, bušić m, rak b, križanović a, kuzmanović elabjer b. optical coherence tomography angiography – a new insight into macular vasculature. seemedj 2019; 3(2); 63-75) seemedj 2019, vol 3, no. 2 optical coherence tomography angiography 64 southeastern european medical journal, 2019; 3(2) introduction optical coherence tomography angiography (oct-a) is a novel non-invasive technology for detailed imaging of retinal and choroidal vasculature of the macular area. it utilizes laser light reflectance off moving erythrocytes, generating information about volumetric blood flow in all retinal layers and the choroid (1). the resolution of oct-a is comparable to the resolution of histological sections (2). unlike fluorescein angiography (fa) or indocyaninegreen angiography (icg), oct-a does not require the use of dye (1). dye-based angiography has been the gold standard for imaging macular and choroidal vasculature for decades. because of the risk of adverse reactions, particularly vomiting, nausea, allergic reaction and anaphylaxis, coupled with the time-consuming nature of the examination (10 to 30 minutes), dye-based angiography is not frequently used for monitoring the course of retinal vascular diseases. while fa can only delineate superficial retinal vasculature, icg imaging is restricted to visualization of the choroidal circulation. moreover, fa is not capable of imaging the radial peripapillary capillary network (rpc), which is very important for monitoring of glaucoma (3). fa is still the gold standard for detecting choroidal (cnv), retinal (nve, neovascularization elsewhere), disc (nvd), neovascularization of the disc) and irideal (nvi, neovascularization of the iris) neovascularization (4). optical coherence tomography angiographycreating an image optical coherence tomography optical coherence tomography (oct) is a diagnostic technique that enables in vivo crosssectional visualization of the tissue in focus. it is currently used for imaging in cardiology, ophthalmology, oncology and dermatology (5). in ophthalmology, it was first introduced in 1991 by huang et al. (6). oct uses interferometry to measure the amplitude and delay of reflected or backscattered near-infrared light from ocular structures. the depth of structure measured in this fashion is known as axial scan (a-scan). by sequential arranging of multiple a-scans in the transverse direction, a b-scan or cross-sectional image is generated (7). oct is based on two techniques: time-domain (td-oct), introduced in 1996 (8), and frequencydomain (fd-oct), devised as spectral-domain (sd-oct) and swept-source (ss-oct). the importance of the advent of fd-oct technology is the acceleration of slow scan so that artefacts caused by motion in earlier td-oct devices are no longer an impediment for better imaging of small vessels (9). doppler oct (doct) was first introduced concurrently by izatt et al. (10) and chen et al. (11) in 1997. doct provides quantitative volumetric information about blood flow, together with vascular and structural anatomy. however, it is limited to larger vessels (12). in clinical practice, doct is not widely used (12, 13). optical coherence tomography angiography (oct-a) oct-a is a three-dimensional functional extension of oct which uses repeated b-scans of the same retinal location to detect blood flow. in 2012, oct-a was introduced as a method for imaging the retinal microvasculature (14). oct-a signals are primarily used to detect the presence or absence of vessels, rather than to provide information about blood flow speed, enabling three-dimensional en face imaging (15). if retinal location is stationary (i.e. if there is no blood flow), the repeated b-scans will be identical. however, if the tissue’s optical scattering is timedependent because of the blood flow through the region, the repeated b-scans will vary. the most important vascular component that can induce backscattering of light is red blood cells. areas of faster blood flow will show greater change over a unit of time. the exact relationship of this change in regard to flow speed depends on many parameters, such as oct beam size and blood vessel size, and is not necessarily linear (16). seemedj 2019, vol 3, no. 2 optical coherence tomography angiography 65 southeastern european medical journal, 2019; 3(2) by using different algorithms, oct-a can monitor flow even in transverse motion, while the other form of monitoring the motion of rbcs, doppler shift (used in doct), can monitor flow only in axial motion. this enables oct-a to monitor flow in microvasculature, while doct can monitor flow only in larger vessels (17). optical resolution of commercially available oct-a systems ranges between 5-10 μm in the axial, and averages ∼20 μm in the transverse direction (16). smaller resolution improves the differentiation of retinal vasculature but increases sensitivity to eye motion. if the signal is below a threshold, a mask is generated. errors in imaging caused by bulk tissue motion are reduced by using different eye tracking modalities (15). oct-a quantification of blood flow, namely flow index and vessel density, has great clinical importance. flow index is the average flow signal in the area of interest. vascular density is the percentage of the area occupied by vessels (18). image artefact is an anomaly in the visual presentation of information derived from an object. projection artefact, one of the most important types of artefacts, represents the appearance of the object at a deeper location than where it exists in reality due to disturbance of the signal. shadowing is the attenuation of a signal behind a scattering opacity or obstruction that absorbs the signal. displacement artefact is caused by eye motion where one part of the image is from one retinal location, while the remaining part of the image is from a different retinal location. stretch artefact is related to software correction of eye motion in which part of the image appears to be stretched. white line artefact is a white line seen due to eye movement (19). one of the most important limitations of oct-a is a fixed area of the central field of view measuring 3 × 3, 6 × 6 and 12 × 12 mm. visualization of the peripheral retina is thus not possible (19). oct-a can detect blood flow above the minimum threshold only. areas with flow under the threshold remain invisible (20, 21). for example, in case of branch retinal vascular occlusion, slow-flow areas may be perceived as areas of non-perfusion (21). moreover, oct-a is incapable of accurately determining vascular leakage, which is especially important in neovascular age-related macular degeneration (namd), diabetic macular oedema, and retinal vein occlusion (22). because of the need for steady fixation, it is difficult to obtain images of children’s retina. clinical use of oct-a in oct-a, vascular abnormalities manifest as abnormal vessel density (dry age-related macular degeneration, amd), anomalous vessel geometry (dilated vessels, aneurysms in diabetic retinopathy, dr), abnormal flow (cnv) and absent flow (nonperfusion/capillary dropout in retinal artery or vein occlusion). therefore, oct-a is currently extensively useful for patients with variable retinal diseases (15). oct-a concurrently obtains images of both retinal and choroidal macular vasculature divided into different layers based on their depth. moreover, oct-a enables the imaging of rpc, originating from choroidal vessels, which is particularly vulnerable to glaucoma and retinal vascular occlusion (23). retinal capillary plexuses are divided into (a) superficial vascular plexus (svp), located in the retinal nerve fibre layer (rnfl) and ganglion cell layer (gcl), and (b) deep vascular plexus (dvp) that extends down to the inner nuclear layer (24). dvp is subdivided into intermediate (icp) and deep capillary plexus (dcp) (25). additionally, oct-a is the only method capable of visualizing cc separately from the deeper choroid (15). foveal avascular zone (faz) the capability of oct-a to image capillaries with high resolution has enabled researchers to study the foveolar avascular zone (faz) in greater detail than it had been possible in the past with fa. on an oct-a scan, faz is presented as a discoid zone within the macula that is devoid of capillaries (figure 1). seemedj 2019, vol 3, no. 2 optical coherence tomography angiography 66 southeastern european medical journal, 2019; 3(2) figure 1. oct-a image of the left eye of a healthy 60-year-old female patient. (a) photography of the macula. (b) foveal anatomy. (c) foveal layers with depicted red blood cells (yellow). (d) red blood cells (yellow) in the foveal area. clear distinction is visible between (e) svp, (f) dvp and (g) avascular complex of the retina. images obtained from spectralis® oct (heidelberg engineering, heidelberg, germany). oct-a-optical coherence angiography, oct-optical coherence tomography, svp-superficial vascular plexus, dvp-deep vascular plexus faz borders are manually drawn on a certain capillary plexus level. parameters defining faz are divided into two groups. area (in mm2), perimeter (length of the faz borders, in mm) and feret’s diameter (maximum diameter of faz, in mm) asses the size, while circularity, axial ratio, roundness and solidity represent the shape of faz. faz circularity is the degree of resemblance to a perfect circle (4π × area/perimeter2). axial ratio is obtained from a best-fit ellipse of the faz (length of major axis/length of minor axis of a best-fit ellipse). roundness uses the best-fit ellipse and is similar to circularity, but is insensitive to irregular borders along the perimeter of the faz (4π × area/(length of major axis)2). solidity describes the extent to which a shape is convex or concave (area/convex area). these parameters are calculated using computer software (26). most studies that investigated faz parameters in healthy individuals reported a larger deep area compared to the superficial area of faz (27, 28, 29, 30). superficial and deep faz area are larger in females (26, 31, 32), and this is possibly related to thinner fovea (32). the faz area and foveal thickness at both the svp and dvp levels exhibited significant inverse correlation (26, 29, 30). this could be explained by an association between higher metabolic demand of a thicker retina and a reduction of the faz area (30). previous studies using fa observed an increase in the faz size with advancing age. however, fa as a diagnostic method is limited when it comes to svp imaging and should not be correlated with oct-a (32, 33). the studies that the examined changes that occur in faz with aging are inconclusive. some studies reported significant (34, 35), while others observed insignificant changes in the faz area with aging (27, 28, 29, 36). yu et al. found an increase in the faz size by 1.48% annually, with a decrease in vascular density by 0.4% (35). in studies using fa, it was proposed that in patients aged 40 or older, age was positively correlated with the faz area (32); however, studies using oct-a did not support this conclusion (36). coscas et al. divided their participants by age into three groups: 20– 39 years old, 40–59 years old, and 60 years old or older (37). the faz size was smaller in the oldest group compared to the two younger seemedj 2019, vol 3, no. 2 optical coherence tomography angiography 67 southeastern european medical journal, 2019; 3(2) groups at the level of svp. no statistically significant difference was found for the level of dvp among the groups (37). a single study evaluated the faz shape in healthy eyes and demonstrated that none of these parameters was significantly correlated with age, sex and refractive error (26). there are no homogenous studies about faz parameters in children (figure 2). figure 2. oct-a image of the right eye of a healthy 4-year-old female patient. (a) photography of the macula. (b) foveal anatomy. (c) foveal layers with depicted red blood cells (yellow). (d) red blood cells (yellow) in the foveal area. clear distinction is visible between (e) svp, (f) dvp and (g) avascular complex of the retina. images obtained from spectralis® oct (heidelberg engineering, heidelberg, germany). oct-a-optical coherence angiography, oct-optical coherence tomography, svp-superficial vascular plexus, dvp-deep vascular plexus choroidal neovascularization (cnv) the advent of oct has enabled new classification of cnv: type 1 (beneath the rpe), which is the most common type, type 2 (above the rpe) (figure 3) and type 3 (intra-retinal). seemedj 2019, vol 3, no. 2 optical coherence tomography angiography 68 southeastern european medical journal, 2019; 3(2) figure 3. oct-a image of the right eye of a 71-year-old male patient with type 2 cnv. (a) photography of the macula with oedema of the papillomacular area. (b) foveal anatomy. (c) foveal layers with depicted red blood cells (yellow). (d) red blood cells (yellow) in the foveal area. oct images demonstrate mild oedema of retinal layers and bruch’s membrane rupture with nv emerging from the choroid. (e) svp devoid of vessels in the area of the pathologic process. (f) dvp with nv. (g) nv emerging from the choroid. images obtained from spectralis® oct (heidelberg engineering, heidelberg, germany). oct-a-optical coherence angiography, cnv-choroidal neovascularization, oct-optical coherence tomography, svp-superficial vascular plexus, dvp-deep vascular plexus, nv-neovascularization type 1 is the most common type of cnv in amd. fa is incapable of determining whether cnv is above or beneath the rpe and thus of defining the type of cnv or of detecting a polyp in polypoidal choroidal vasculopathy, a subtype of type 1 cnv. cnv therefore requires multimodal imaging (oct, fa, icg) (38). oct-a can vastly improve the definition of exact cnv dimensions compared to fa (39, 40, 41, 42). this is of high importance as larger cnvs have poorer visual outcome (39). oct-a introduced new biomarkers for predicting disease activity and duration. greater vessel calibres are fairly unresponsive to treatment due to excessive covering with pericytes (39). encouraged by these insights, the greatest vascular calibre (gvc) was proposed as the marker of long-standing disease. the gvc could reveal the duration of cnv, which is important due to excessive damage caused by long-standing type 1 cnv, which remains asymptomatic longer than type 2 (39). more mature vessels are found in type 1 cnv compared to type 2 (43). as a biomarker for active cnv, tiny branching vessels (tbv) can be used due to their presence in 82% of active lesions and only 30% of quiescent ones (44). the peripheral arcade is also present in 82% of active lesions compared to 40% of quiescent ones (44, 45). tbvs are important because they are more vulnerable to treatment due to their lack of pericytes compared to prominent vessels. in addition, pericytes appear later than angiogenesis, which means that tbvs could be related to the exudative status of the lesion; they could enable prompt treatment in order to preserve the macular architecture and thus, visual acuity (45). in light of these findings, it seems reasonable to include oct-a imaging in the monitoring of all patients with cnv (45). nonexudative (subclinical) cnv was first described in post-mortem eyes by green et al. seemedj 2019, vol 3, no. 2 optical coherence tomography angiography 69 southeastern european medical journal, 2019; 3(2) (46) and sarks et al. (47) as abnormal choroidal vessels passing through breaks in bruch’s membrane in the absence of overlying haemorrhage or exudation. these lesions, which presented as plaques on icg (48), can now be diagnosed more accurately using oct-a (49). compared with icga, the sensitivity and specificity of oct-a in detecting subclinical cnv was reported as 81.8% and 100 %, respectively (50). it would be beneficial to determine the presence of subclinical cnv in fellow eyes of patients with unilateral exudative amd, which ranges from 6.25% to 27%, respectively (49). the progression rate of subclinical cnv to exudative form is 20% and the existence of a possible protective effect of subclinical cnv against geographic atrophy progression has been suggested (51). reduction in cc flow adjacent to cnv could be a marker of imminent exudation in subclinical cnv, as new evidence demonstrates (49, 51). exudation may be triggered by the underlying progression of cc nonperfusion. hypoxia of the retinal pigment epithelium causes the release of abnormal vascular endothelial growth factor (vegf) signalling with growth and eventual exudations of a cnv (49). glaucoma pathophysiology of glaucoma and the onset of changes in macular vasculature are yet to be elucidated. recent studies have suggested that macular vascular changes in glaucoma may be related to mechanisms other than intraocular pressure (iop) (52, 53). in early glaucoma, oct-a revealed focal loss of rpc (54) and decreased parafoveal vascular density (52). in eyes with central visual field defects, faz perimeter could be used as a biomarker for detecting glaucoma (55). in a recent study, eyes with open-angle glaucoma demonstrating central visual field defects (cvfds) confined to a single hemifield exhibited a larger faz area and a less circular faz than those with peripheral visual field defects (55). loss of faz circularity and increased size of the faz area were significantly correlated with the presence and severity of cvfd at initial presentation (55). parafoveal and peripapillary vascular density decrease in primary open-angle glaucoma compared to normal tension glaucoma (52, 53) is inconsistent with the earlier described mechanism of normal tension glaucoma development through its association with vascular compromise as a contrast to primary open-angle glaucoma, where the pathophysiology is mostly correlated with intraocular pressure (56). it is unclear whether vascular density changes in glaucoma antedate ganglion cell loss or are a direct result of loss of neural tissue and thus a marker for both primary open-angle glaucoma and normal tension glaucoma (53). diabetic retinopathy optical coherence tomography angiography offers a non-invasive alternative in the investigation of diabetic retinopathy. faz has been one of the most extensively investigated areas in diabetic retinopathy (57). in eyes with diabetic retinopathy, the circularity and axial ratio of the faz are significantly different from normal eyes. these metrics could be predictors of disease progression and response to therapy (58). however, even without retinopathy, oct-a demonstrated significantly enlarged faz areas compared to controls – in both the scp and dcp (57). in eyes with diabetic retinopathy, oct-a can identify microaneurysms (ma), microvascular abnormalities associated with diabetic macular oedema (dmo), and areas of capillary nonperfusion associated with neovascularization, allowing enhanced analysis compared to fa in that their intraretinal location beyond svp can be identified (59). however, the sensitivity for ma detection and small field of view are currently the major limitations of oct-a technology (57). a lower number of ma visible on oct-a as compared to fa may be due to slower flow speeds in ma that are beyond the oct-a detection threshold (60) or due to focal staining of vessel walls allowing superior identification by fa (61). in eyes with proliferative diabetic retinopathy, oct-a can visualize preretinal seemedj 2019, vol 3, no. 2 optical coherence tomography angiography 70 southeastern european medical journal, 2019; 3(2) neovascularization. compared to fa, oct-a has demonstrated moderate agreement for grading of diabetic macular ischemia (59). by enabling three-dimensional visualization of the individual retinal vascular networks, oct-a is enhancing our understanding of the role of deeper vasculature in the pathogenesis of diabetic retinopathy and maculopathy. oct-a can differentiate between different subgroups of diabetic retinopathy severity by measuring perfusion indices in eyes with dr and branching complexity of vessels (57). the decrease in vascular density with the progression of the disease has already been established (62, 63). reduction of vascular density in dvp occurs earlier in the course of the disease and this finding gives rise to possible new studies concerning vascular density in dvp as a marker of disease severity in earlier stages of diabetic retinopathy (60). faz enlargement and reduction of parafoveal deep and superficial vascular density can be beneficial as a marker of increased disease severity in diabetic retinopathy (60). central serous chorioretinopathy diagnosing cnv as a complication of chronic central serous chorioretinopathy (cscr) using fa is difficult due to the confusing signs of the primary disease, such as choroidal hyperpermeability, retinal pigment epithelium leakage, or atrophy and cystic macular oedema (64). oct-a has the advantage of identifying only the flow, without the exudative component, and it allows for depth-correlated visualization of flow with separation of the signal generated from the pathologic area between rpe and the bruch membrane from the cc. these advantages of oct-a are more prominent for type 1 cnv (65). cnv locations correspond to slightly irregular and hyperreflective rpe areas (66), which is consistent with earlier observations (67). with oct-a, a higher detection rate of cnv (mainly type 1) in chronic cscr is achieved compared to icg and fa (65). thus, in case of cscr, using oct-a b-scan and en-face mode is always recommended in order to define the area of flat irregular pigment epithelial detachment, which is claimed to be imperative for the cnv diagnosis (65). ocular oncology a significant enlargement of the deep faz and a decrease in the superficial and deep vascular density in eyes with choroidal melanoma were observed compared to healthy eyes (68). these changes are correlated with larger tumour size and presence of subretinal fluid, which could elucidate the pathogenesis of vision loss in patients with melanoma (68). one possible mechanism behind these changes is vegfinduced microvascular compromise preceding macular oedema (68). these features are absent in eyes with choroidal nevi, giving rise to easier differentiation of small melanoma from choroidal nevi (68). early vascular changes in radiation retinopathy that can be detected by oct-a as irregular widening of faz, discontinuity of retinal vasculature and retinal ma have been used as a part of a new grading scheme and treatment decisions for radiation retinopathy (69). conclusion oct-a is an important new non-invasive tool for imaging of the chorioretinal vasculature. it has provided new insights into the pathogenesis of multiple retinal and choroidal diseases, but its full contribution is yet to come. regarding the paediatric population, the normal values have to be defined first in order for us to accurately elucidate the pathology. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. seemedj 2020, vol 4, no. 1 stigmatization of patients with viral infections and mass psychogenic illness 71 southeastern european medical journal, 2020; 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4(1) original article levels of ldl cholesterol, triglyceride and urate in patients with type 2 diabetes mellitus 1 dunja šojat* 1, 2, marko pirić 1, 2, marja klarić 1, matej šapina 1, 3, zvonimir popović 1, 3, tatjana bačun 1, 3 1 faculty of medicine, josip juraj strossmayer university of osijek, croatia 2 healthcare centre osijek, croatia 3 clinical hospital centre osijek, croatia *corresponding author: dunja šojat, dunja.sojat@gmail.com received: mar 1, 2020; revised version accepted: apr 2, 2020; published: apr 27, 2020 keywords: type 2 diabetes, ldl cholesterol, triglycerides, urates abstract aim: the study aimed to examine ldl cholesterol, triglyceride and urate levels in patients of both sexes with type 2 diabetes mellitus (dm2t) in family medicine offices and to examine whether there is a difference in these parameters between obese patients and patients with normal body weight with dm2t, and between patients with and without manifest cardiovascular diseases. participants and methods: the study was organized as a cross-sectional study. it included 136 participants of both sexes diagnosed with dm2t, who were divided into groups of patients with or without adiposity and into groups of patients with or without experience of cardiovascular event. general and demographic data were collected, as well as data on experiencing cardiovascular events and levels of ldl cholesterol, triglycerides and urates. results: the average ldl cholesterol level was 2.93 mmol/l, the average triglyceride level was 1.65 mmol/l and the average urate level was 326.36 µmol/l. only 12.5% of participants reached target ldl cholesterol levels, while levels of triglycerides and urates were within recommended limits. 24.3% of participants had experienced cardiovascular events and 39.7% of participants were obese. there was no significant difference in levels of ldl cholesterol, triglycerides and urates in participants who had experienced a cardiovascular event and those who had not. there was a significantly higher concentration of triglycerides in obese patients than in patients with normal body weight (p = 0.005). conclusion: in addition to regulation of glycaemia in patients with dm2t, statin doses should be increased in order to reach the target levels of ldl cholesterol. when it comes to obese patients, education courses on physical activity and diet should be conducted more often and, if necessary, fibrates should be included in therapy in order to reduce additional cardiovascular risks. (šojat* d, pirić m, klarić m, šapina m, popović z, bačun t. levels of ldl cholesterol, triglyceride and urate in patients with type 2 diabetes mellitus. seemedj 2020; 4(1); 32-39) seemedj 2020, vol 4, no. 1 ldl cholesterol, triglyceride and urate with type 2 diabetes mellitus 33 southeastern european medical journal, 2020; 4(1) introduction diabetes mellitus is a chronic and progressive disease. nowadays it is one of the most important public health problems and for many years it has been among the top ten causes of death in the republic of croatia (1). the international diabetes federation estimates that more than 420 million people worldwide suffer from diabetes, and it is predicted that in the next 25 years, this number will reach 630 million (2). the number has increased every year in the republic of croatia and persons diagnosed with diabetes, currently counting over 260,000 people, are considered to be only half of the actual total number of patients (3). despite the good-quality and affordable healthcare in the republic of croatia, diabetes is often detected or treatment starts in an advanced stage, which affects both the quality and the cost of diabetes treatment. croatian health insurance fund data for 2016 showed that the total cost of treating diabetes in the republic of croatia amounted to hrk 4.6 billion, accounting for almost 20% of the total croatian health insurance fund budget (4). pathophysiology of diabetes is complex and it includes the interaction of genetic and environmental factors (1). modern, sedentary lifestyle and excess energy intake undoubtedly lead to a state of overweight and obesity, where, due to the increase of visceral fat tissue, insulin resistance occurs; this is, in addition to beta-cell dysfunction and impaired insulin secretion, a major pathophysiological disorder for development of diabetes, along with many other conditions, such as increased gluconeogenesis, increased glucagon secretion in pancreatic αcells, decreased incretin effect, increased lipolysis, increased renal glucose reabsorption and neurotransmitter disorder (1, 5). for this reason, educating patients on physical activity and diet planning plays an important role in the long-term regulation of diabetes (6). it is estimated that 75% of diabetic patients suffer from at least one other associated disease. cardiovascular diseases occur 15 years earlier in such patients, being the leading cause of mortality (7). diabetic patients have a higher prevalence of coronary heart disease, a more severe degree of coronary ischemia and 2 to 3 times more frequent myocardial infarction with a worse prognosis (8). factors identified for the development of atherosclerosis, which we can affect, are hyperlipoproteinemia, hyperglycaemia, hypertension, hyperhomocysteinemia, smoking and obesity. it has been shown that by controlling these factors, cardiovascular mortality is significantly reduced, even in the case of patients with type 2 diabetes (9, 10). according to the cardiovascular risks table referred to in the 2019 guidelines, patients with type 2 diabetes belong to the group of moderate cardiovascular risk if they are under 50 years of age and the disease itself has been present for less than 10 years without additional risk factors. in case of type 2 diabetes lasting for 10 years or longer with the presence of a risk factor or type 2 diabetes without damage to the target organs, cardiovascular risk was assessed as high. diabetic patients who suffered damage to target organs (e.g., proteinuria or severe chronic kidney disease with glomerular filtration rate – egfr < 30 ml/min/1.73 m2, retinopathy or neuropathy) or had at least 3 risk factors belong to the group of patients with very high cardiovascular risk. the target ldl cholesterol level for the group of patients with very high cardiovascular risk is < 1.4 mmol/l and a 50% or greater decrease of the initial level, while target ldl cholesterol levels at the time when this research was conducted, according to the 2016 guidelines, amounted to < 1.8 mmol/l or a 50% or greater decrease of the initial level of ldl cholesterol (11). despite the available therapy, diabetic patients are still at additional cardiovascular risk regarding the rapid development of atherosclerosis. although the prevalence of increased levels of ldl cholesterol is lower each day, the prevalence of high levels of triglycerides and low levels of hdl cholesterol has doubled in recent years, which may be connected to the increasing number of obese people (metabolic syndrome). therefore, body mass regulation and pharmacotherapy for lowering triglyceride levels and increasing hdl cholesterol levels play an important role (12). seemedj 2020, vol 4, no. 1 ldl cholesterol, triglyceride and urate with type 2 diabetes mellitus 34 southeastern european medical journal, 2020; 4(1) the objectives of this research were to determine the average concentrations of ldl cholesterol, triglycerides and urates in patients of both sexes with type 2 diabetes in family medicine offices of the healthcare centre osijek and to examine whether there is a difference in these parameters between obese patients and patients with normal body weight, as well as between patients with and without manifest cardiovascular disease. the research also examined whether obese diabetic patients and diabetic patients with manifest cardiovascular disease would reach target levels of ldl cholesterol, triglycerides and urates. participants and methods the research was organized as cross-sectional, with data collected at a defined time, and it was conducted in two family medicine offices in the healthcare centre osijek. before the research, participants were provided with detailed information on the planned research and they read and signed the informed consent document. participants were over 18 years of age, of both sexes, diagnosed with type 2 diabetes. a total of 136 participants were included in the research, of which 55 were men and 81 were women, with an average age of 69.33 (sd = 10.87). the above-mentioned selection of participants and the size of the sample ensured the representativeness of the sample and objective results. participants were anonymised, with each participant being assigned a unique code, i.e. a number. for the purpose of the research, the participants were further divided into groups of patients who have experienced a cardiovascular event and those who have not, as well as into groups of obese patients and patients with normal body weight. the research was approved by the ethics committee of the healthcare centre osijek and the ethics committee of the faculty of medicine osijek of the josip juraj strossmayer university of osijek. when visiting the family medicine office, after the participants had been informed of the contents of the informed consent document and after they had signed it, which was a prerequisite for participating in the research, the following data were collected: demographic data (sex, age), data on duration of diabetes, data on body height and weight, and whether the occurrence of a cardiovascular event (acute myocardial infarction, angina pectoris, stroke, transient ischemic attack) was documented in the available medical records. data on the existence of diabetes-related complications (diabetic retinopathy, nephropathy and macrovascular complications) were also collected from the medical records. data on concentrations of ldl cholesterol, triglycerides, urates, plasma glucose and hba1c were collected from laboratory findings. ldl cholesterol, triglyceride and urate concentrations were measured using the beckman coulter olympus au680 chemistry analyser in accordance with the manufacturer’s instructions. triglyceride concentration was measured photometrically, using glycerolphosphate-oxidase (gpo-pap), ldl cholesterol concentration was measured using a homogeneous enzymatic assay, and urate concentration was determined by photometry using the uv uricase method. concentrations of glycated haemoglobin were determined using the turbidimetric immunoinhibition method, whereby the sample for analysis was whole blood with edta as an anticoagulant. statistical analysis categorical data were presented as absolute and relative frequencies. numerical data were described as the arithmetic mean and standard deviation in case of normal distribution and as the median and interquartile range in other cases. differences between categorical variables were tested using the χ2 test and fisher’s exact test, where necessary. normality of distribution of numerical variables between two independent groups was tested using student’s t-test and mann-whitney u test in case of deviation from normal distribution. correlation between the variables was expressed as pearson’s correlation coefficient in cases where variables follow a normal distribution or spearman’s correlation coefficient in cases where variables do not follow a normal distribution. all p values are two-tailed. the level of statistical significance was set at α = 0.05. the medcalc statistical software version 18.11.3 was used for statistical analysis. seemedj 2020, vol 4, no. 1 ldl cholesterol, triglyceride and urate with type 2 diabetes mellitus 35 southeastern european medical journal, 2020; 4(1) results the research included 136 participants, of which 81 were women and 55 were men. the average duration of diabetes in patients was 9 (4-14) years. the mean level of fasting plasma glucose test was 8.43 ± 2.99 mmol/l and the mean level of hba1c was 7.15% with interquartile range of 6.70-8.30%. 97 (71.3%) participants experienced diabetes-related complications and the most common complication was diabetic retinopathy, with 16.2% of participants experiencing it. 15.4% of participants had an acute myocardial infarction and 12.5% of participants had a stroke or a transient ischemic attack. 14.0% of participants experienced two or more complications. 24.3% of participants were diagnosed with cardiovascular diseases, and 39.7% were obese (bmi > 30 kg/m2). there was no statistically significant difference with regard to adiposity (chi-square test, p = 0.084) and incidence of cardiovascular diseases (chi-square test, p = 0.060) between men and women. in the highest number of participants, 64%, ldl cholesterol levels were > 2.5 mmol/l and only 12.5% of participants reached the recommended target levels of ldl cholesterol (< 1.8 mmol/l). mean triglyceride and urate levels were within the recommended levels and no statistically significant difference was found between men and women (table 1).. table 1. mean levels of ldl cholesterol, urates and triglycerides in patients with type 2 diabetes level ldl cholesterol (mmol/l) 2.93 ± 1.02 triglycerides (mmol/l) 1.65 (1.25 – 2.40) urates (mmol/l) 326.36 ± 98.55 the difference in levels of ldl cholesterol, triglycerides and urates was also examined between the group of participants who have experienced a cardiovascular event and the group of participants who have not experienced it, and no statistically significant difference was found. target ldl cholesterol levels were not reached in either group, target triglyceride levels were reached in the group of participants who have not experienced a cardiovascular event, and target urate levels were reached in both groups (table 2). table 2. mean levels of ldl cholesterol, triglycerides and urates in patients with type 2 diabetes – comparison of the group of participants who have experienced a cardiovascular event and the group of participants who have not experienced cv event (n = 33) not experienced cv event (n = 103) p ldl cholesterol (mmol/l) 2.88 ± 1,06 2.95 ± 1.01 0.733* triglycerides (mmol/l) 1.80 (1.30 – 2.45) 1.60 (1.20 – 2.40) 0.650** urates (mmol/l) 332.79 ± 11.02 324.28 ± 9.51 0.668* *student’s t-test; **mann-whitney u test finally, our research examined the difference in ldl cholesterol, triglyceride and urate levels in obese patients and patients with normal body weight. the mean triglyceride level was significantly higher in the group of obese patients than in the group of patients with normal body weight (mann-whitney u test; p = 0.005), while the tested difference in ldl seemedj 2020, vol 4, no. 1 ldl cholesterol, triglyceride and urate with type 2 diabetes mellitus 36 southeastern european medical journal, 2020; 4(1) cholesterol and urate levels was not significant. participants with normal body weight did not reach target ldl cholesterol levels, while obese participants did not reach target ldl cholesterol, triglyceride or urate levels (table 3). table 3. mean levels of ldl cholesterol, triglycerides and urates in patients with type 2 diabetes – comparison of obese participants and participants with normal body weight obese (n = 54) normal body weight (n = 82) p ldl cholesterol (mmol/l) 3.05 ± 0.92 2.86 ± 1.08 0.325* triglycerides (mmol/l) 2.20 (1.48 – 2.75) 1.50 (1.10 – 2.08) 0.005** urates (mmol/l) 342.13 ± 13.85 316.48 ± 10.64 0.141* *student’s t-test; **mann-whitney u test discussion one of the biggest challenges in family medicine is regular and in-depth monitoring of a chronic patient’s medical condition, especially of patients with type 2 diabetes and patients with multimorbidities, as well as continuous education on the importance of maintaining normal glucose levels, physical activity and diet planning in order to prevent the development of microvascular and macrovascular complications, which increase patient mortality as well as reduce the quality of life. factors which often complicate regular monitoring and counselling of chronic patients, including diabetic patients, in family medicine offices are the increasing number of patients visiting out-patient clinics per day and their lack of response due to insufficient levels of education. all of the above shows that providing healthcare for patients with type 2 diabetes is a difficult task and a special challenge, as well as that treatment priorities should be set together with the patients, bearing in mind the professional guidelines, associated diseases, existing therapy and the patients’ social status. guidelines of the american diabetes society (ada) and the european association for the study of diabetes (easd) issued in october 2018 recommend hba1c levels < 7% for most patients, ≤ 6.5% for younger patients and patients without comorbidities, and ≤ 7.5% for older patients with longer duration of disease and comorbidities (13). mean hba1c levels in our research were 7.15%, which corresponds to the guidelines’ recommendations, given that our participants were older patients with multimorbidities whose average age was 69.33 ± 10.87. in a similar largescale study conducted in family medicine offices in the republic of croatia, hba1c levels were 7.6% on average, which is slightly above the recommended levels (14). the most common complication in participants is diabetic retinopathy, which 16.2% of participants had been diagnosed with; this can be compared to the results obtained in croatian and worldwide studies, with values ranging between 18 and 20% (14,15). the average urate and triglyceride levels in participants were within the reference values irrespective of sex. only 12.5% of participants reached target ldl cholesterol levels, and ldl cholesterol levels of 64% of participants were over 2.5 mmol/l, which also corresponds to results obtained in large-scale studies conducted at the level of primary healthcare worldwide, where ldl cholesterol levels of 60 to 65% of participants amounted to 2.6 mmol/l and over (16). the average triglyceride level was 1.65 (1.25 – 2.40) mmol/l, which is a better result than the one obtained in other studies conducted in family medicine offices in the republic of croatia. research conducted by naqvi syeda et al. in 2017 showed a strong correlation between high triglyceride levels and high hba1c levels in diabetic patients (r = 0.278, p value < 0.0001). given the results, the use of hba1c levels as seemedj 2020, vol 4, no. 1 ldl cholesterol, triglyceride and urate with type 2 diabetes mellitus 37 southeastern european medical journal, 2020; 4(1) markers for dyslipidaemia, especially for hypertriglyceridemia, has been proposed in order to minimize cardiovascular risks in diabetic patients through timely administration of adequate therapy (14,17). average urate levels in our research were also within the reference values, 318.97 ± 11.11 µmol/l for women and 337.44 ± 13.09 µmol/l for men. urates or uric acid are organic substances produced as a final product of purine metabolism. hyperuricemia is a condition in which plasma urate levels exceed recommended levels and it is frequently found in persons with congestive heart failure. given that persons with type 2 diabetes are at increased risk for the development of cardiovascular diseases, and ultimately heart failure, it is considered that correction of hyperuricemia is very important for them. many studies examined the correlation between increased urate concentration and the development of insulin resistance and diabetes, dyslipidaemia, arterial hypertension and abdominal obesity, but the results were contradictory (18, 19, 20). the average body mass index of our participants was 29.48 ± 5.64 kg/m2, with 39.7% of participants having a body mass index > 30 kg/m2, which classifies them as obese. tested differences in obesity between men and women were not significant (chi-square test, p = 0.084). in croatia, approximately 25.3% of men and 34.1% of women are considered obese, which is consistent with the results of our research. nowadays, obesity is almost the biggest health problem as it has become a pandemic, and in pathophysiological terms it is closely linked to the onset of many chronic diseases, including diabetes and cardiovascular diseases. there are many causes of obesity and the healthcare system’s attempt to stop this pandemic has not produced satisfactory results so far. programs in many countries are focused mainly on therapeutic rather than preventive approach, since prevention requires serious long-term investments with generally uncertain results. prevention programs should focus on children and young people in order for them to adopt healthy habits and behaviour patterns in a timely manner, which, in the long run, would certainly contribute to reducing obesity in adults, and thus to decreasing its negative effects on population health (21). the research compared ldl cholesterol, triglyceride and urate levels between the groups of obese diabetic patients and diabetic patients with normal body weight. as expected, ldl cholesterol, triglyceride and urate levels were higher in the group of obese patients, but a statistically significant difference between the two groups was only shown with regard to triglyceride levels (mann-whitney u test, p = 0.005). the group of obese patients did not reach the target levels of any of the examined parameters (ldl cholesterol, triglycerides and urates), while the group of patients with normal body weight reached the target levels of urate and triglycerides, confirming the correlation between adiposity and these parameters. cardiovascular diseases are considered to be a major cause of mortality in patients with type 2 diabetes, but also in the general population. many studies have demonstrated the existence of equal mortality risk caused by coronary diseases in patients with type 2 diabetes who have not experienced a cardiovascular event and patients who are not suffering from type 2 diabetes, but who have experienced a cardiovascular event (22). diabetic patients who had an acute myocardial infarction are 43% more likely to experience a recurrent cardiovascular event than diabetic patients who have never experienced a cardiovascular event (23). out of the total number of our participants, 24.3% of them have experienced a cardiovascular event and no statistical difference was found between men and women (chi-square test, p = 0.060), which is very likely connected to the high average age of participants, i.e. menopause in women, which makes cardiovascular risk equal for both sexes. a large prospective korean study examined the correlation between ldl cholesterol levels and the onset of cardiovascular events in patients with type 2 diabetes who had not experienced a cardiovascular event. the patients were monitored for 7 years. the average ldl cholesterol levels in participants were 2.94 seemedj 2020, vol 4, no. 1 ldl cholesterol, triglyceride and urate with type 2 diabetes mellitus 38 southeastern european medical journal, 2020; 4(1) mmol/l, which is similar to the results obtained in our research. the patients were further divided into groups of patients who were taking statin therapy and those who were not. there was a statistically significant increased risk of cardiovascular event occurrence in the group of patients who were not taking statin therapy, while ldl cholesterol levels were > 3.37 mmol/l and > 1.8 mmol/l in the group of diabetic patients taking statin therapy (24). target ldl cholesterol levels in our research were not reached in either the group of patients who have experienced a cardiovascular event or the group who have not experienced it, whereas target triglyceride levels were only reached in the group of patients who have not experienced a cardiovascular event. despite the use of statin and antihypertensive therapy in corresponding doses and adequate glucoregulation, diabetic patients are at additional cardiovascular risk due to increased triglyceride levels and decreased hdl cholesterol levels, which is why it is extremely important to regulate hdl cholesterol and plasma triglyceride levels in addition to ldl cholesterol levels, as well as to observe urate levels for possible worsening of the cardiovascular condition that would further contribute to microand macrocirculatory damage. given the poor ldl cholesterol levels shown, it can be concluded that it is of great importance for patients with type 2 diabetes to regularly check their lipid profile and to increase statin doses, if necessary, in order to reach the target ldl cholesterol levels. in cases of triglyceridemia, fibrate therapy should be considered in addition to statin therapy. nevertheless, the most important thing is to educate patients and administer nonpharmacological therapy for regulating body weight, as well as to encourage physical activity, which will, in the long term, have a positive effect on reducing triglyceride and urate levels, as well as on glucoregulation in patients with type 2 diabetes. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. .references 1. topić e, primorac d, janković s, štefanocić m. et al. medicinska biokemija i laboratorijska medicina. zagreb 2nd edition. medicinska naklada: 2018. 2. international diabetes federation. diabetes atlas – 9th edition. available at: https://www.diabetesatlas.org/en/. accessed: 22 february 2020. 3. croatian institute of public health. crodiab registar. available at: https://www.hzjz.hr/sluzba-epidemiologijaprevencija-nezaraznih-bolesti/crodiabregistar/. accessed: 22 february 2020. 4. croatian institute of public health. usporedba pokazatelja o vodećim javnozdravstvenim problemima u republici hrvatskoj i europskoj uniji. available at: https://www.hzjz.hr/wpcontent/uploads/2017/01/pokazatelji_rh_eu. pdf. accessed: 22 february 2020. 5. defronzo ra. from the triumvirate to the ominous octet: a new pradigm for the treatment of type 2 diabetes mellitus. diabetes. 2009; 58:773-95. 6. evert ab, boucher jl, cypress m, dunbar sa, franz mj, mayer-davis ej, neumiller jj, nwankwo r, verdi cl, urbanski p, yancy ws jr. nutrition therapy recommendations for the management of adults with diabetes. diabetes care. 2013; 36(11):3821-42. 7. low wang cc, hess cn, hiatt wr, goldfine ab. clinical update: cardiovascular disease in diabetes mellitus: atherosclerotic cardiovascular disease and heart failure in type 2 diabetes mellitus mechanisms, management, and clinical considerations. circulation. 2016; 133(24):2459-502. 8. nesto r.w. prevalence of and risk factors for coronary heart disease in diabetes mellitus. seemedj 2020, vol 4, no. 1 ldl cholesterol, triglyceride and urate with type 2 diabetes mellitus 39 southeastern european medical journal, 2020; 4(1) up to date. available at: https://www.uptodate.com/contents/prevale nce-of-and-risk-factors-for-coronary-heartdisease-in-diabetes-mellitus. accessed: 22 february 2020. 9. turner rc, millns h, neil haw, stratton im, manley se, matthews dr, holman r. risk factors for coronary artery disease in non-insulin dependent diabetes mellitus: united kingdom prospective diabetes study. bmj. 1998; 316(7134):823-28. 10. gamulin s. marušić m. kovač z et al. patofiziologija. zagreb 7th edition. medicinska naklada: 2011. 11. mach f, baigent c, catapano al, koskinas kc, casula m, badimon l chapman mj, de backer gg, delgado v, ference ba, graham im, halliday a, landmesser u, mihaylova b, pedersen tr, riccardi g, richter dj, sabatine ms, taskinen m-r, tokgozoglu l, wiklund o, esc scientific document group. 2019 esc/eas guidelines for the management of dyslipidaemias: lipid modification to reduce cardiovascular risk: the task force for the management of dyslipidaemias of the european society of cardiology (esc) and european atherosclerosis society (eas). european heart journal, 2020; 41(1):111–188 https://doi.org/10.1093/eurheartj/ehz455 12. krstačić g. rezidualni rizik-danas, sutra...;još jedan pogled na rezultate accord studije i podstudija. kardio list. 2010; 5(12):294-8. 13. davies mj, d’alessio da, fradkin j, kernan wn, mathieu c, mingrone g, rossing p, tsapas a, wexler dj, buse jb. management of hyperglycemia in type 2 diabetes, 2018. a consensus report by the american diabetes association (ada) and the european association for the study of diabetes (easd). diabetes care. 2018; 41(12):2669-701. 14. bralić lang v, bergman marković b, kranjčević k. family physician clinical inertia in glycemic control among patients with type 2 diabetes. med sci monit. 2015; 21:403-411. 15. deshpande ad, harris-hayes m, schootman m. epidemiology of diabetes and diabetes-related complications. phys ther. 2008; 88(11):1254–64. 16. mozaffarian d. dietary and policy priorities for cardiovascular disease, diabetes, and obesity: a comprehensive review. cirkulation. 2016; 133(2):187-225. 17. naqvi s, naveed s, ali s, ahmad sm, khan ra, raj h, shariff s, rupareliya c, zahra f, khan s. correlation between glycated hemoglobin and triglyceride level in type 2 diabetes mellitus. cureus. 2017; 9(6):1347. 18. pušeljić s, milas v. hiperuricemija i hipouricemija – klinički značaj, dijagnostički i terapijski postupci. pediatr croat. 2009; 53(1):178185. 19. zjačić-rotkvić v, katalinić d, berković m. metabolička inzulinska rezistencija i metabolizam purina. medicus. 2004; 13(2):51-6. 20. butković m. mokraćna kiselina kao mogući čimbenik bolesti srca i bubrega. acta med croatica. 2016; 70:233-39. 21. medanić d, pucarin-cvetković j. pretilost – javnozdravstveni problem i izazov. acta med croatica, 2012; 66:347-55. 22. bertoluci mc, rocha vz. cardiovascular risk assessment in patients with diabetes. diabetol metab syndr. 2017;9: 25. 23. bulugahapitiya u, siyambalapitiya s, sithole j, idris i. diabet med. 2009; 26(2):142-8. 24. kim mk, han k, joung hn, baek kh, song kh, kwon hs. cholesterol levels and development of cardiovascular disease in koreans with type 2 diabetes mellitus and without pre-existing cardiovascular disease. cardiovasc diabetol. 2019; 18(139). doi: 10.1186/s12933-019-0943-9. seemedj 2020, vol 4, no. 2 improvement of monitoring of hcv patients in croatia 28 southeastern european medical journal, 2020; 4(2) original article the need for systematic monitoring and improved surveillance of hepatitis c patients in croatia 1 leona radmanić 1*, nataša cetinić balent 1, petra šimičić 1, adriana vince 1,2, snježana židovec lepej 1, oktavija đaković rode 1,3 1 university hospital for infectious diseases „dr. fran mihaljević“, zagreb, croatia 2 school of medicine, university of zagreb, croatia 3 school of dental medicine, university of zagreb, croatia *corresponding author: leona radmanić, leona.radmanic@gmail.com received: mar 12, 2020; revised version accepted: mar 12, 2020; published: nov 12, 2020 keywords: hepatitis c virus, monitoring, diagnosis abstract aim: the aim of this study was emphasizing the need for a more systematic monitoring of patients diagnosed with hcv in croatia. methods: from 2014 to 2018, at the university hospital for infectious diseases, sera from 23,524 patients were tested for hcv. confirmatory testing was performed by western blot. adult patients with an anti-hcv positive screening test were analysed. hcv rna was quantified by real-time pcr, while hcv genotypes and subtypes were determined by pcr and the reverse hybridization method. results: a total of 428 anti-hcv elisa-positive adults were analysed (68.7% males, 31.3% females, median age 43 years, range 19-88 years). hepatitis c was confirmed by wb in 390, while 28 patients had borderline wb results. anti-hcv was not confirmed by wb in 10 patients. hcv rna was tested in 331 patients and viremia was detected in 218 patients. there was no data on hcv rna in 97 patients (22.66%). hcv genotypes/subtypes were determined in 185 of 218 anti-hcv wb positive patients. genotype 1 was detected in 97/185 (52.43%), genotype 2 was detected in 3/185 (1.62%), while subtype 3a was detected in 76/185 (41.08%) and genotype 4 in 9/185 patients (4.86%). conclusion: in a five-year period, the hcv seroprevalence rate in subjects tested at the university hospital for infectious diseases was 1.81%. according to the data analysed, almost one quarter of patients with detected anti-hcv antibodies were not treated further, which indicates the need for a systematic monitoring of patients diagnosed with hcv. it is necessary to determine viremia after a positive anti-hcv screening result in order to initiate treatment and prevent hcv-related complications. (radmanić l, cetinić balent n, šimičić p, vince a, židovec lepej s, đaković rode o. the need for systematic monitoring and improved surveillance of hepatitis c patients in croatia. seemedj 2020; 4(2); 28-34) seemedj 2020, vol 4, no. 2 improvement of monitoring of hcv patients in croatia 29 southeastern european medical journal, 2020; 4(2) introduction both a general practitioner and a secondarycare specialist are involved in the diagnosis of chronic viral hepatitis and in the clinical management of infected patients [1]. the initial diagnosis and management of chronic hepatitis relies on primary-care physicians to identify and screen patients who were in contact with hepatitis c, since most people with chronic hepatitis are asymptomatic until the development of cirrhosis or hepatocellular carcinoma [2]. the who recommends education of primary care physicians about the risks factors of acquiring hcv infection and encourages increased activity in finding new patients in screening programs. approximately 71 million people worldwide have chronic hepatitis c, of which 15 million in europe, and about 400,000 people in the world die from the effects of hepatitis c per year. croatia is a country with a low incidence rate of hepatitis c. it is estimated that 40,000 people are anti-hcv positive in croatia. in may 2016, the world health assembly endorsed the global health sector strategy on viral hepatitis, which proposed to eliminate viral hepatitis as a public health threat by 2030. elimination of viral hepatitis as a public health threat requires 90% reduction in incidence and 65% reduction in mortality in comparison with the 2015 baseline, along with the improvement of viral hepatitis diagnostic coverage up to 90% and treatment of 80% of eligible patients [4]. to reach these targets, the who regional office for europe is encouraging member states to plan and strengthen national responses to viral hepatitis through awareness-raising, surveillance, prevention, strengthening of laboratory capacity and provision of guidance on testing and treatment [5]. implementation of the global health sector strategy would prevent 7.1 million deaths between 2015 and 2030 [4]. hepatitis c virus (hcv) diagnostics starts with determination of anti-hcv antibodies [3,6]. each positive anti-hcv screening result requires further hcv rna detection in order to confirm actual contact with hcv in the past. if hcv rna is negative, positive anti-hcv results should be confirmed by western immunoblotting (wb) [7,8]. in 2019, who recommended offering treatment with direct acting antivirals (daa) to all individuals diagnosed with hcv infection who are 12 years of age or older, irrespective of disease stage (with the exception of pregnant women) [9]. priority for treatment is defined by the stage of fibrosis, the risk of progression to advanced disease, the presence of extrahepatic manifestations and comorbidities, patients with hbv or hiv coinfection and patients with indication for organ transplantation [10]. the aim of this study was emphasizing the need for a systematic monitoring of patients diagnosed with hcv. this must include the determination of viremia after a positive antihcv screening result in order to administer a corresponding therapy since, according to the data analysed, almost one quarter of patients with detected anti-hcv antibodies are not treated further. patients and methods from 2014 to 2018, at the university hospital for infectious diseases in zagreb, sera from 23,524 patients were tested for anti-hcv antibodies by using either enzyme immunoassay (elisa) for simultaneous detection of anti-hcv antibodies and capsid antigen (hcv ag-ab) or only antihcv antibody determination (biorad, france). as required, each of the 428 positive elisa sera was confirmed by western blot (inno-lia hcv score fujirebio; recomline hcv igg mikrogen). hcv rna was quantified by real-time pcr (abbott realtime hcv) and hcv genotypes and subtypes were determined by pcr and the reverse hybridization (inno-lipa hcv genotyping) method. results this study included 428 anti-hcv elisa-positive adults who were newly diagnosed in the period from 2014 to 2018 and over 18 years of age. median age of patients was 43 years, ranging from 19 to 88 years. regarding gender, 68.7% of seemedj 2020, vol 4, no. 2 improvement of monitoring of hcv patients in croatia 30 southeastern european medical journal, 2020; 4(2) the patients were male and 31.3% were female. according to the available diagnoses from the referral, 293 (293/428; 68.45%) patients had chronic hepatitis or elevated hepatic lesion, 52 (52/428; 12.14%) patients were injecting drug users and 83 (83/428; 19.39%) patients had other diagnoses, for example, neurological and muscular diseases and factors affecting the health system. anti-hcv was confirmed by western blot in 390 patients (91.12%), while 28 patients (6.54%) had borderline western blot results. when it comes to borderline western blot results, negative result was confirmed with molecular testing in 16 patients, hcv infection was excluded in two patients with paired sera and infection status was unknown in 10 patients. positive anti-hcv elisa was not confirmed by western blot in 10 patients (2.34%). molecular testing for hcv rna was performed in 331 patients – 65.86% (218/331) of the patients had measurable viremia (table 1). table 1. results of western blot and hcv rna in anti-hcv (elisa) positive patients tested at uhid anti-hcv hcv rna patients n (%) enzyme-linked immunoassay western blot positive positive positive 218 (65.86) negative 87 (26.28) positive borderline negative 16 (4.83) positive negative negative 10 (3.02) total 331 (100.00) median viremia was 456.024.5 iu/ml (range 7843.041.938 iu rna hcv/ml). hcv rna was not detected in 87 patients with positive hcv wb, 16 with borderline and 10 with negative hcv wb. there was no data on hcv rna testing at the uhid for 85 patients with positive and 12 with borderline hcv wb (97/428; 22.66%) (table 2). table 2. results of western blot and hcv rna in anti-hcv (elisa) positive patients anti-hcv hcv rna patients n (%) enzyme-linked immunoassay western blot positive positive positive 218 (50.93) negative 87 (20.33) unknown 85 (19.86) positive borderline negative 16 (3.74) unknown 12 (2.80) positive negative negative 10 (2.34) total 428 (100.00) seemedj 2020, vol 4, no. 2 improvement of monitoring of hcv patients in croatia 31 southeastern european medical journal, 2020; 4(2) according to the available diagnoses from the referral, 48 of 97 patients with positive anti-hcv elisa and unknown viremia were patients who had chronic hepatitis or elevated hepatic lesion (49.48%). seventeen of 97 patients were injecting drug users (17.52%) and 32 of 97 patients had other diagnoses, for example, neurological and muscular diseases and factors affecting the health system (32.99%). hcv genotypes and subtypes were determined in 185 of 218 anti-hcv wb positive patients with hcv rna >1.000 iu/ml in the serum. genotype 1 was detected in 97 (97/185; 52.43%) patients, genotype 2 in 3 (3/185; 1.62%) patients, subtype 3a in 76 (76/185; 41.08%) /185 and genotype 4 in 9 (9/185; 4.86%) patients. subtypes 1a and 1b of genotype 1 were further distinguished. subtype 1a was detected in 57 (57/97; 58.76%) patients and subtype 1b in 36 (36/97; 37.11%) patients. in four patients, the subtype of genotype 1 could not be determined (figure 1). figure 1. hcv genotype/subtype distribution in hcv rna-positive patients (n = 185) discussion hcv prevalence estimates range from 0.4% to 5.2% in the world, with countries in the north and west of europe having lower estimates (0.9%) than countries in the east of europe (3.3%) [11,12]. in a five-year period, hcv seroprevalence rate in subjects diagnosed at the uhid was 1.81%, which was slightly higher than the estimate (0.8-1.0%) for the croatian population [13]. however, most of the data on prevalence was obtained through the serological testing of samples from voluntary blood donors, who were a strictly controlled group. hcv prevalence in blood donors continuously declined from 1.38% in 1992 to 0.0009% in the last decade. therefore, it is to be expected that the actual prevalence in the general population is higher [13,14]. nevertheless, this data suggests that hcv seroprevalence in croatia is most similar to the seroprevalence rate in western european countries. according to the latest analysis on the distribution of hcv genotypes and subtypes in croatia, croatian patients were mostly infected with hcv genotype 1 (56.63%), followed by seemedj 2020, vol 4, no. 2 improvement of monitoring of hcv patients in croatia 32 southeastern european medical journal, 2020; 4(2) genotype 3 (37.23%), genotype 4 (4.21%) and genotype 2 (1.83%). this is very similar to our data, according to which genotype 1 was detected in 52.43% patients, genotype 2 in 1.62% patients, subtype 3a in 41.08% and genotype 4 in 4.86% patients [15]. however, genotype 1 subtyping showed 58.76% of subtype 1a infections and 37.11% subtype 1b infections in our study, while national studies suggest a higher prevalence of subtype 1b in croatia [15,16]. our cohort study tested 23,524 patients for antihcv antibodies to show how many adults with a positive anti-hcv screening result were accurately diagnosed, including determination of viremia, in the period from 2014 to 2018. epidemiological and clinical data from the referrals and the uhid database suggest that 218 of 331 patients who were molecularly tested at the uhid had measurable viremia, while there was no data for 97 anti-hcv-positive patients. this suggests the possibility they were tested at another institution or that they were simply not treated further. this indicates the need for a more systematic monitoring and determination of viremia in potential patients in order to initiate treatment. according to the available diagnoses from the referral, 17.50% of the patients were people who injected drugs and, probably due to their lifestyle, were lost to secondary specialist care. furthermore, 49.50% of the patients were people who had chronic hepatitis or elevated hepatic lesion and 33% of the patients were people with other diagnoses, for example, neurological and muscular diseases and factors affecting the health system. since injecting drug use is one of the most efficient routes for hcv transmission, there is a very high prevalence of hcv in people who inject drugs in most european countries, while in croatia it ranges from 29% to 65%, depending on the geographical region [13]. according to becchini et al., general practitioners’ role and referral back to primary care vary within and between countries – most general practitioners are rarely involved in monitoring clinical outcomes other than some side effects among patients undergoing antiviral treatment [1,8]. a lack of uniform practice suggests that in some patients with serologically confirmed presence of the infection, additional nucleic acid testing may not always be performed. therefore, the role of the general practitioner and specialists involved in clinical management of chronically infected patients should be clarified in order to ensure that the patients are followed-up and accurately diagnosed [1]. the croatian reference centre for diagnostics and treatment of viral hepatitis performs initial check-ups of patients with acute and chronic hepatitis b and hepatitis c infection, serological and molecular testing to detect parameters of virus infection, pretreatment evaluation of the patients (liver biopsy, fibroscan) along with treatment and monitoring of patients after the end of treatment. national reference centre also provides strategies and guidelines for optimization of diagnostics and standardization of treatment of viral hepatitises [17]. however, in croatia, there is no national hepatitis treatment registry used by all physicians prescribing daas and patients are diagnosed and treated at various institutions across the country. such decentralization enables easier access to care for patients, whereas an early diagnosis and successful treatment not only prevent hcv-related complications, but also stop infectiousness. on the other hand, it is possible that the monitoring of patients and overview of the treatment process is better in specialized, centralized settings than in unspecialized, decentralized settings [18]. conclusion it is necessary to improve the monitoring and surveillance of hepatitis c patients in croatia since almost one quarter of patients with detected anti-hcv antibodies are not treated further. determination of viremia after positive anti-hcv screening would ensure adequate treatment of the patients and prevent hcvrelated complications. acknowledgement. none. seemedj 2020, vol 4, no. 2 improvement of monitoring of hcv patients in croatia 33 southeastern european medical journal, 2020; 4(2) disclosure funding. this publication was supported by the grant “strengthening the capacity of cervirvac for research in virus immunology and vaccinology”, kk.01.1.1.01.0006, awarded to the scientific centre of excellence for virus immunology and vaccines and co-financed by the european regional development fund. competing interests. none to declare. references 1. bechini a, levi m, falla a, ahmad a, veldhuijzen i, tiscione e, bonanni p. the role of the general practitioner in the screening and clinical management of chronic viral hepatitis in six eu countries. j prev med hyg 2016; 57(2):e51– e60. 2. ferrante jm, winston dg, chen ph, de la torre an. family physicians’ knowledge and screening of chronic hepatitis and liver cancer. fam med 2008; 40(5):345-51. 3. https://www.hzjz.hr/aktualnosti/virusnihepatitisi/ 4. world health organization, combating hepatitis b and c to reach elimination by 2030, https://apps.who.int/iris/bitstream/handle/10 665/206453/who_hiv_2016.04_eng.pdf?sequ ence=1 (2016), accessed date: november 2019. 5. who regional office for europe, hepatitis c in the who european region, http://www.euro.who.int/en/healthtopics/communicablediseases/hepatitis/data-andstatistics/factsheet-hepatitis-c-in-the-who-european-region1 (2019), accessed date: november 2018. 6. cetinić balent n, mikulić r, đaković rode o. imunoenzimski test za određivanje protutijela na pojedinačne antigene virusa hepatitisa c kao potvrdni test u dijagnostici hepatitisa c. infektološki glasnik 2013; 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2010. available from: http://ecdc.europa.eu/en/publications/public ations/ter_100914_hep_b_c%20_eu_neighbo urhood.pdf. 13. vilibić čavlek t, kučinar j, kaić b, vilibić m, pandak n, barbić lj, stevanović v, vraneš j. epidemiology of hepatitis c in croatia in the european context. world journal of gastroenterology 2015; 21(32):9476-93. 14. civljak r, kljakovic-gaspic m, kaic b, bradaric n. viral hepatitis in croatia. viral hepat j 2014; 20:49-56. 15. vince a, židovec lepej s, bingulacpopović j, miletić m, kuret s, sardelić s., baćavrakela i., kurelac i. distribution of hepatitis c virus genotypes and subtypes in croatia 20082015, cent, eur, j, publ, health 2018; 26(3):159–63. 16. vince a., iščić-beš j., židovec lepej s., baća-vrakela i., bradarić n., kurelac i., vince db. distribution of hepatitis c virus genotypes in croatia – a 10 years retrospective study of four seemedj 2020, vol 4, no. 2 improvement of monitoring of hcv patients in croatia 34 southeastern european medical journal, 2020; 4(2) geographic regions, coll antropol 2006; 30(suppl 2):139–43. 17. reference centers of ministry of health in croatia http://www.bfm.hr/en_gb/page/referentnicentri (2020). accessed date: january 2020. bregenzer a, conen a, knuchel j, friedl a, eingenmann f, näf m, ackle p, roth m, fux ca. management of hepatitis c in decentralized versus centralized drug substitution programmes and minimally invasive point-ofcare tests to close gaps in the hcv cascade. swiss med wkly 2017; 147:w14544. i author contribution: acquisition of data: radmanić l, cetinić balent n, šimičić p, vince a, židovec lepej s, đaković rode o administrative, technical or logistic support: radmanić l, cetinić balent n, šimičić p, vince a, židovec lepej s, đaković rode o analysis and interpretation of data: radmanić l, cetinić balent n, šimičić p, vince a, židovec lepej s, đaković rode o conception and design: radmanić l, cetinić balent n, šimičić p, vince a, židovec lepej s, đaković rode o critical revision of the article for important intellectual content: radmanić l, cetinić balent n, šimičić p, vince a, židovec lepej s, đaković rode o i drafting of the article: radmanić l, cetinić balent n, šimičić p, vince a, židovec lepej s, đaković rode o final approval of the article: radmanić l, cetinić balent n, šimičić p, vince a, židovec lepej s, đaković rode o guarantor of the study: radmanić l, cetinić balent n, šimičić p, vince a, židovec lepej s, đaković rode o obtaining funding: radmanić l, cetinić balent n, šimičić p, vince a, židovec lepej s, đaković rode o provision of study materials or patients: radmanić l, cetinić balent n, šimičić p, vince a, židovec lepej s, đaković rode o statistical expertise: radmanić l, cetinić balent n, šimičić p, vince a, židovec lepej s, đaković rode o seemedj 2020, vol 4, no. 2 patient satisfaction in zenica-doboj canton 129 southeastern european medical journal, 2020; 4(2) original article a study of patient satisfaction with healthcare in zenica-doboj canton 1 šeila cilović lagarija *1, elma kuduzović 2, nino hasanica 2, sead begagić 2, amela džubur-alić 3, delila lisica 3 1 public health institute of the federation of bosnia and herzegovina, sarajevo, bosnia and herzegovina, 2 institute for health and food safety zenica, zenica, bosnia and herzegovina, 3 faculty of medicine, university of sarajevo, sarajevo, bosnia and herzegovina *corresponding author: šeila cilović lagarija, seila.cilovic@gmail.com received: apr 7, 2020; revised version accepted: jul 16, 2020; published: nov 12, 2020 keywords: patient satisfaction, healthcare, europep abstract aim: patient satisfaction and patient evaluation of healthcare can be seen as important results of provided care, as they reflect the level to which the patient’s subjective and objective needs have been met. the aim was to evaluate patient satisfaction with healthcare and compare the results for 2017 with the results from 2011. methods: the study was conducted in the territory of zenica-doboj canton in may 2011 and in october 2017. a questionnaire was filled out by 2,008 examinees in 2011 and by 2,000 examinees in 2017 outside healthcare institutions (in the street), using the europep questionnaire as a research instrument. the average age of the respondents was 38.4. the participants in the study were 52 % male and 48 % female. student’s t-test was used to compare the results between the two samples. results: a statistically significant difference was observed between the mean patient satisfaction in 2011 (3.19 ± 0.3, min. = 2.6, max. = 3.83, p = 0.00032) and the mean patient satisfaction in 2017 (3.47 ± 0.17, min. = 3.14, max. = 3.94, p = 0.000647), t(23) = 3.75. increase in patient satisfaction in 2017 is evident compared to their satisfaction in 2011. conclusion: surveying the satisfaction of healthcare recipients should be a common method of work as it gives the patients the impression that their opinion is valued, while at the same time it indicates to healthcare staff that their attitudes may need to be changed, their knowledge expanded and the organization of work improved, if they want to provide services of improved quality. (cilović lagarija š, kuduzović e, hasanica n, begagić s, džubur-alić a, lisica d. a study of patient satisfaction with healthcare in zenica-doboj canton seemedj 2020; 4(2); 129-135) seemedj 2020, vol 4, no. 2 patient satisfaction in zenica-doboj canton 130 southeastern european medical journal, 2020; 4(2) introduction the patient is the one to be questioned whether the provided health care has helped to improve their health or quality of life. it is not only the results of healthcare that matter in terms of improved health; it is also important how the healthcare is provided: access to care, organization of services, staff education and their communication with the patient are all relevant factors. there is increased awareness among persons with health insurance policies and healthcare staff that patients can and must have a central role in defining optimal care and improved quality of healthcare. engagement of patients in the process of improving healthcare is not only desirable; it is also a social, economic and technical necessity (1). new concepts, such as the central role of patients, strengthening of patients, patients as partners and joint decisions, demonstrate an emancipation of patients. engagement of patients is not only important from the ethical perspective. it is important to investigate not only the expectations, needs and priorities of patients within general healthcare practices, but also to gather information about the experiences of patients provided with care. most patients can provide more than an opinion and assessment of care and treatment they received from a doctor or other healthcare staff. such information may be very important. it may help the practitioners select those aspects of care where improvement is necessary. on the other hand, patient satisfaction and their evaluation of care may be observed as important results of provided care, since they reflect the level to which their subjective and objective needs are met. this cannot be observed as the only relevant result, as sometimes the patients have unrealistic demands; however, patient satisfaction can generally be seen as an important addition to other types of measures (health status, quality of life or costs) required to evaluate the quality of general medical practice (2). information about the patient’s evaluation of care is mainly collected through (written) questionnaires filled out by patients who go to see a doctor or get the questionnaire by mail. in principle, patient surveys are among the most popular methods of assuring quality in a healthcare system. material and methods the europep instrument has been created to provide feedback to general practitioners / family doctors, general practitioner’s offices as well as organizations of patients/healthcare recipients. it is a standardized instrument for evaluating patient satisfaction with healthcare. for this study, the europep questionnaire was translated into bosnian (3). the study was conducted in the territory of zenica-doboj canton in may 2011 and in october 2017. the questionnaire was filled out by 2,008 examinees in 2011 and by 2,000 examinees in 2017 outside healthcare institutions (in the street). student’s t-test was used to compare the results between the two samples. results the study was conducted in the territory of zenica-doboj canton for the purpose of evaluating satisfaction with healthcare. the europep methodology (23 questions with a 5point answering scale), which was developed by an international consortium of researchers and general practitioners in 1995, was used. the research also used a modified form with questions relevant for our field of work (length of time spent waiting for an examination, evaluation of staff actions during the stay in a medical institution, payment of services and medication, and reason for payment). the research was conducted in may 2011 and in october 2017. the survey was performed on a sample of 2,000 respondents outside medical institutions (in the street) in order to get as many objective answers as possible. the aim of the research was to obtain results for 2017 and compare them with those from 2011 (4,5). seemedj 2020, vol 4, no. 2 patient satisfaction in zenica-doboj canton 131 southeastern european medical journal, 2020; 4(2) the average age of the respondents was 38.4 (age range 12-83). the study included 51.95% of male participants and 48.05% of female participants. the respondents’ education structure is shown in table 1, indicating that the highest number of respondents had secondary school education. figure 1 shows the distribution of respondents by municipalities. figure 1. structure of respondents per municipality in 2017 table 1. level of respondents’ education in 2017 level of education no. 10 without primary school 13 primary school 257 secondary school 1.230 college 250 university 240 total 2.000 table 2 provides a classification of the respondents from zenica-doboj canton, showing their occupation in numbers and percentages. according to the occupation classification, the highest number of respondents were employed in the public sector (23.55%). table 2. respondents’ occupation in 2017 occupation no. % unknown 16 0.8 student 315 15.7 5 unemployed 221 11.05 private entrepreneur 92 4.6 employed in the public sector 268 13.4 employed in the private sector 471 23.5 pensioner 204 10.2 farmer 76 3.8 freelance (artist) 43 2,15 housewife 259 12.9 5 other 35 1.75 total 2.00 0 100 by comparing the results of the average grade for questions in table 3 for the period 2011–2017, we obtained the following results: by using an 29 0 88 46 19 39 0 19 87 73 480 3444 39 115 70 44 198 24 39 112 137 143 121 0 100 200 300 400 500 600 grad selo seemedj 2020, vol 4, no. 2 patient satisfaction in zenica-doboj canton 132 southeastern european medical journal, 2020; 4(2) independent samples t-test, comparison of the results for 2011 and 2017 was made for patient satisfaction in regard to provided services and treatment by medical staff. a statistically significant difference was noted between the mean patient satisfaction in 2011 (3.19 ± 0.3, min = 2.6, max = 3.83, p = 0.00032) and the mean patient satisfaction in 2017 (3.47 ± 0.17, min = 3.14, max = 3.94, p = 0.000647), t(23) = 3.75. the difference between mean values per group (mean difference of 0.41) was small (eta squared = 0.0468). table 3. average grade per europep question (on a scale of 1 to 5 for each question) average grade per europep question do you think that the doctor spends sufficient time with you? 3.33 does the doctor show any interest in your problem? 3.41 do you feel better when you tell the doctor about your problem? 3.46 does the doctor involve you in making a decision about your treatment? 3.36 does the doctor listen to you carefully while you are presenting your problems? 3.52 does the doctor provide you with all information about your diseases? 3.42 does the doctor try hard to relieve your symptoms as soon as possible? 3.47 does the doctor help you feel better and return to your everyday work? 3.49 does the doctor perform a physical examination? 3.75 does the doctor perform a detailed physical examination? 3.51 does the doctor work on the prevention of various diseases? 3.46 does the doctor explain why you need to undergo additional tests and analyses? 3.44 does the doctor provide explanation about your symptoms and disease? 3.47 does the doctor help with your emotional problems related to your health condition? 3.30 does the doctor explain to you why it is important to comply with his/her instructions? 3.46 does the doctor explain what he/she is doing during the examination? 3.43 does the doctor explain what you can expect at a specialist examination in hospital? 3.36 are you assisted by other medical staff (nurse at the clinic)? 3.56 can you make an appointment with the doctor? 3.80 is it easy to make a phone call to the doctor? 3.31 can you seek advice from the doctor by phone? 3.14 do you wait long in the waiting room? 3.46 does the doctor respond fast in emergency situations? 3.94 discussion by comparing surveys conducted in bosnia and herzegovina and other countries from the region, we reached the following conclusions: a survey conducted at the healthcare centre of zenica in 2013 showed that patient satisfaction with general practice offices was statistically seemedj 2020, vol 4, no. 2 patient satisfaction in zenica-doboj canton 133 southeastern european medical journal, 2020; 4(2) most significant with regard to making appointments with practitioners at a time suitable for the patients (p = 16.28), the possibility of making a phone call to the clinic (p = 32.55), and the length of time spent in the waiting room (p = 30.42). obtained data confirmed a high level of patient satisfaction with units of general practice in primary healthcare. the europep questionnaire seems to be a useful tool for reviewing patient satisfaction with medical services (6). in comparison to this study, in bulgaria, 58.7% of respondents on average rated the level of care received as excellent. the waiting time in the waiting room was the most poorly rated item (33.8%). the item “keeping patients’ records and data confidential” was the most highly rated item (88.8%). patients were less satisfied with “providing quick services for urgent health problems” (78.5% excellent or good) and “getting an appointment to suit them” (76.2% excellent or good) (7). in 2016, a cross-sectional study was performed among visitors of the emergency medical service (ems) of gorenjska, slovenia. it took into account the waiting time, which is considered to be associated with the success of the ems organizational model. the europep questionnaire was used for rating the degree of patient satisfaction. results showed a score higher than 4. patients were least satisfied with the length of time spent waiting for an examination. research results confirmed that the effectiveness of the ems organizational model had an impact on the degree of patient satisfaction (8). this study showed that most respondents had a positive opinion about the behavior of gps, emphasizing their gentleness during medical examinations (83%), respect for privacy (82%), as well as a benevolent attitude towards patients (77%). however, despite the positive assessment of their behavior, in the opinion of 52% of the respondents, doctors did not offer them any preventive examinations, and in many cases (43%) did not provide information about a healthy lifestyle. a third of the patients (32%) was not informed about the side effects of medication by a doctor. the results showed that fewer than half of the gps were interested in the psychosocial sphere of their patients. only 27% of the respondents received their doctor’s help in regard to dealing with fears about their health. an even lower percentage of respondents stated that their doctor expressed interest in their personal (23%) or material (23%) situation, while 35% of patients received questions from their doctors about family members (9). in the research conducted in zenica-doboj canton in 2015, the questionnaire was made on the basis of the europep standardized questionnaire regarding patient satisfaction with healthcare. the older population evaluated secondary and tertiary healthcare practices better, they needed more time to reach health facilities, and they waited longer to receive healthcare services in primary healthcare practice (10). research conducted across turkey’s 81 provinces over the period 2010-2012, using the european patients evaluate general/family practice (europep), showed that the family medicine model significantly improved patient satisfaction across a range of dimensions (11-12). this study has shown great similarity with the results of this research, showing evident increase in patient satisfaction. numerous studies conducted in the area of the federation of bosnia and herzegovina have shown significant improvement and increase in patient satisfaction with healthcare (6, 13-17). conclusion as expected, the analysis of data obtained in the study of patient satisfaction in the area of zenica-doboj canton shows that there was a significant increase in satisfaction in 2017 in comparison with 2011. surveying the satisfaction of healthcare recipients should be a common method of work as it gives the patients the impression that their opinion is important and serves as an indication for medical staff in terms of required changes of attitudes, expansion of knowledge and organization of work. seemedj 2020, vol 4, no. 2 patient satisfaction in zenica-doboj canton 134 southeastern european medical journal, 2020; 4(2) acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare.i references 1. guadagnoli e, ward p. patient participation in decision-making. soc sci med 1998; 47(3):329-39. 2. grol r, wensing m, mainz j, jung hp, ferreira p, hearnshaw h, hjortdahl p, olesen f, reis s, ribacke m, szecsenyi j. european task force on patient evaluations of general practice care (europep). patients in europe evaluate general practice care: an international comparison. br j gen pract 2000; 50(460);882-7. 3. grol r, wensing m. patients evaluate general/family practice. the europep instrument. mediagroep kun/umc, 2000. 4. kantonalni zavod za javno zdarvstvo zenica. izvještaj o zdravstvenom stanju stanovništva i organizaciji zdravstva na području zeničko-dobojskog kantona u 2011. godini. zenica, 2012. 5. institut za zdravlje i sigurnot hrane. informacija o zdravstvenom stanju stanovništva i organizaciji zdravstva na područiju zeničkodobjskog kantona u 2017. godini. zenica, 2019. 6. gavran l, jašarević e, hasanica n. patient satisfaction with primary care: are there differences between the approaches in family and general medicine? med glas 2013; 10(2):37984. 7. dimova r, stoyanova r, keskino d. the europep questionnaire for patient's evaluation of general practice care: bulgarian experience. croat med j 2017; 58(1):63-74. 8. jaklič tk. analysis of patient satisfaction with emergency medical services. open med (wars) 2018; 13:493-502. 9. plentara r, knyszyńska a, bazydło m, zabielska p, kim a, kotwas a, rotter i, kamola j, karakiewicz b. patient satisfaction measure of the quality of primary health care. pomeranian j life sci 2015; 61(3):335-40. 10. spasojević n, hrabač b, huseinagić s. patient's satisfaction with health care: a questionnaire study of different aspects of care. mater sociomed 2015; 27(4):220-4. 11. aktürk z, ateşoğlu d, çiftçi e. patient satisfaction with family practice in turkey: three-year trend from 2010 to 2012. eur j gen pract 2015; 21(4):238-45. 12. sparkes sp, atun r, bӓrnighausen t. the impact of the family medicine model on patient satisfaction in turkey: panel analysis with province fixed effects. plos one 2019;14(1). (ahead of print) 13. mašić i. service quality in health care. in: mašić i, ed. family medicine. sarajevo: avicena, 2007: 95-114. 14. toromanović s. user satisfaction with primary health care as a parameter for assessing quality. sarajevo: university of sarajevo, 2002. master’s thesis. 15. toromanović s, mašić i, novo a. measuring the quality of primary health care: methodology, variables, techniques, assessment. aim 2004; 12:11-6. 16. mašić i, nikšić d. quality and quality assurance in health care. med arh 2003; 57:18996. toromanović s. research of the quality of primary care by measuring user satisfaction of health services. sarajevo: university of sarajevo, 2005. ph.d. dissertation thesis. seemedj 2020, vol 4, no. 2 patient satisfaction in zenica-doboj canton 135 southeastern european medical journal, 2020; 4(2) i author contribution. acquisition of data: cilović lagarija š, kuduzović e, hasanica n, begagić s, džubur-alić a, lisica d administrative, technical or logistic support: cilović lagarija š, kuduzović e, hasanica n, begagić s, džubur-alić a, lisica d analysis and interpretation of data: cilović lagarija š, kuduzović e, hasanica n, begagić s, džubur-alić a, lisica d conception and design: cilović lagarija š, kuduzović e, hasanica n, begagić s, džubur-alić a, lisica d critical revision of the article for important intellectual content: cilović lagarija š, kuduzović e, hasanica n, begagić s, džubur-alić a, lisica d drafting of the article: cilović lagarija š, kuduzović e, hasanica n, begagić s, džubur-alić a, lisica d final approval of the article: cilović lagarija š, kuduzović e, hasanica n, begagić s, džubur-alić a, lisica d guarantor of the study: cilović lagarija š, kuduzović e, hasanica n, begagić s, džubur-alić a, lisica d provision of study materials or patients: cilović lagarija š, kuduzović e, hasanica n, begagić s, džubur-alić a, lisica d statistical expertise: cilović lagarija š, kuduzović e, hasanica n, begagić s, džubur-alić a, lisica d seemedj 2020, vol 4, no. 2 neuroimaging in juvenile headaches 69 southeastern european medical journal, 2020; 4(2) original article validity of neuroimaging in juvenile headaches 1 ivana serdarušić *1, silvija pušeljić 1,2, višnja tomac 1, matea romić 3 1 department of pediatrics, university hospital center osijek, croatia 2 faculty of medicine, josip juraj strossmayer university of osijek, osijek, croatia 3 institute of emergency medicine of osijek-baranja county, osijek, croatia *corresponding author: ivana serdarušić, lega.serdarusic@gmail.com received: mar 1, 2020; revised version accepted: jul 27, 2020; published: nov 12, 2020 keywords: headache, children, neuroimaging abstract aim: the objectives of this study are to evaluate the incidence of headache considering the type of headache, to define the types of headaches, to determine the difference in the status of diagnostic scanning in children with headaches, to establish a correlation between the psychological profile of the child and the incidence of headache, and to establish a correlation between socio-demographic characteristics and the type of headache. patients and methods: the study included 139 patients with headache symptoms up to the age of 18, hospitalized at the pediatric clinic of the university hospital center osijek from 1/1/2017 to 31/12/2018. the data included demographic data, diagnosis, environmental factors, eeg findings, neuroimaging data processing and other indicated medical tests. results: a headache usually occurs between the ages of 12 and 18 (69.8%). it is more common in girls (70.5%). the common localizations are frontal and occipital. altered standard eeg findings were reported in 26.7% of patients. additional neuroradiological processing (brain mri) was indicated in 98 patients (70.5%), with changes found in 56 patients (57.1%). psychological assessment indicated that patients with functional headaches predominantly suffer from anxiety, emotional instability and somatization, while patients with organic headaches reported high stress levels (82%). conclusion: headaches occur more frequently in pubescent girls. the most common concomitant symptoms include nausea and vomiting, while the most common localization is frontal. patients also report emotional instability, cognitive deficits and somatization, as well as high stress levels. headache as a result of psychological tension is the most common diagnosis in the observed group of patients. (serdarušić i, pušeljić s, tomac v, romić m. validity of neuroimaging in juvenile headaches. seemedj 2020; 4(2); 69-76) seemedj 2020, vol 4, no. 2 neuroimaging in juvenile headaches 70 southeastern european medical journal, 2020; 4(2) introduction a headache in childhood is not a rare symptom, regardless of whether the headache background is organic or functional. according to the experience of numerous authors, childhood headache is most commonly of organic origin (1). the headache is determined by localization (frontal, occipital, temporal), pain type (dull, pulsating pain), frequency, duration and associated symptoms and signs (2). the incidence of headaches depends on age and gender, as well as on the type or category of headache. the prevalence of headaches of different types at the age of 7 is 31 51%, increasing to 57-82% at the age of 15 (3). in school children, the most common type are primary headaches, which are defined as headaches that are not associated with an underlying disorder (group 1-4, according to the classification of the international headache society (ihs classification)) and classified as migraines or tension-type headaches (tth). the most common causes of secondary headaches are viral respiratory infections (29-39%) and mild head injuries (4, 5). researchers have found that the incidence of migraine (except for migraine with aura) is similar in girls and boys, while tension-type headaches occur more often in girls, and that gender has some effect on ihs criteria for migraine, but it has almost no effect on those for tension-type headaches (6). risk factors for headaches or chronic headaches are as follows: dysfunctional family situation, regular alcohol consumption, caffeine intake, smoking, low levels of physical activity, physical or emotional abuse, bullying, misconduct at school, and insufficient leisure time (4). each child with a headache requires an individual approach. the assessment of a child suffering from a headache begins with a thorough physical examination, including blood pressure measurement, a neurological examination and a meticulous medical history (3). if a child with recurrent headaches has a normal neurological status during the examination, it is not necessary to routinely (urgently) perform a neuroradiological assessment (brain ct or mri). a neuroradiological assessment is certainly indicated for determining the cause of secondary headaches. cerebrospinal fluid analysis is needed in febrile immunocompromised children with a headache due to suspected meningitis. it is not necessary to routinely perform electroencephalography (eeg) in the evaluation of children with headaches. eeg is indicated if epileptic seizure or a secondary headache (caused by a tumor, head trauma, encephalitis, intracranial hemorrhage or ischemia) is suspected. however, changes in eeg are in principle neither specific nor pathognomonic for specific causes of secondary headaches (7). the purpose of this study was to examine the types of headaches, the most common symptoms of headaches and their comorbidities, and to emphasize psychological examination in addition to the usual neurological treatment. patients and methods the study was conducted at the pediatric clinic of the university hospital center osijek. the study included 139 pediatric patients diagnosed with headaches, hospitalized and treated between 1 january 2017 and 31 december 2018. data from the medical records and information system of the pediatric clinic included: age, sex, headache diagnosis, perinatal risk factors and psychomotor development of the child, child’s admission status (clinical and neurological status), family history, data on the child’s education and/or kindergarten attendance, child’s psychological profile, eeg findings, neuroimaging processing, and other indicated tests. ethical approval for the study was obtained from ethical committee faculty of medicine university of osijek and approval to conduct study was obtained from clinical hospital centre osijek. seemedj 2020, vol 4, no. 2 neuroimaging in juvenile headaches 71 southeastern european medical journal, 2020; 4(2) statistical analysis the data collected were processed using statistical software r (version 3.3.2, www.rproject.org). categorical data are descriptively represented by absolute and relative frequencies. differences between categorical variables were examined using the χ2 test and the binominal test (23, 24). the level of statistical significance was determined as p < 0.05. results the total number of hospitalized patients over the two observed years was 5554, of which 139 had headaches. thus, the prevalence of hospitalization for patients with headaches was 2.5% with a 95% confidence interval of 2.11% to 2.95%. during the two observed years, there were no significant differences in the number of hospitalized patients (p = 0.799). in 2017, the number of hospitalized patients with headaches was 71, and in 2018, it was 68. a significant difference was found in the number of hospitalizations based on gender (p < 0.001). there was a significantly higher proportion of hospitalized female patients (70.5 %) in comparison to male patients (29.5%). when it comes to age, the highest prevalence of hospitalized patients was between the ages of 12 and 18 (69.78 %), followed by those between the ages of 5 and 12 (26.62 %), while only five patients were under the age of 5 (table 1). table 1. patients’ demographic data n (%) p* year 2017 71 (51.08) 0.799 2018 68 (48.92) gender male 41 (29.5) < 0.001 female 98 (70.5) age group ≤ 5 5 (3.6) < 0.001 5 to 12 37 (26.62) 12 to 18 97 (69.78) *χ2 test there was a significantly higher proportion of newly diagnosed headaches (59 %), p = 0.034. in regard to the number of hospitalizations, a significant number of patients were hospitalized more than once (58.7%). functional headaches were reported in 41.01% of cases and organic headaches with comorbidities in 58.99% of cases (p = 0.034) (table 2). table 2. related characteristics of hospitalized patients with headaches n (%) p* headache newly diagnosed 82 (58.99) 0.034 controlled 57 (41.01) number of hospitalizations 1 57 (41.3) 0.041 > 2 81 (58.7) headache type functional 57 (41.01) 0.034 organic 82 (58.99) *χ2 test the highest number of patients complained of frontal headaches (40.29%), followed by occipital (17.27 %), parietal (13.67 %) and temporal (10.79 %) headaches. a lower number of patients suffered from headaches with multiple localizations: frontotemporal – 4.32 %, parieto-occipital – 2.16 % and diffuse – 3.6 % (table 3). table 3. headache localization n (%) p* frontal 56 (40.29) < 0.001 parietal 19 (13.67) occipital 24 (17.27) temporal 15 (10.79) frontotemporal 6 (4.32) parieto-occipital 3 (2.16) diffuse 5 (3.6) *χ2 test a statistically significant difference was found in the frequency of concomitant symptoms. nausea and vomiting (44.85 %) are reported most frequently, followed by dizziness (33.09 %), loss seemedj 2020, vol 4, no. 2 neuroimaging in juvenile headaches 72 southeastern european medical journal, 2020; 4(2) of consciousness (25.74 %), blurred vision (25 %), weakness and fatigue (17.65 %), tingling in hands (11.51 %). tics are rarely reported as a concomitant symptom of headache (0.74 %) (table 4). table 4. concomitant symptoms of headaches n (%) p* nausea and vomiting 61 (44.85) < 0.001 dizziness 45 (33.09) loss of consciousness 35 (25.74) blurred vision 34 (25) febrility 6 (4.41) tingling sensation in hands 16 (11.51) absence seizure (petit mal) 5 (3.68) photophobia 13 (9.56) hand tremors 4 (2.94) weakness and fatigue 24 (17.65) tics 1 (0.74) non-epileptic seizure 13 (9.56) *χ2 test there were 80 % of patients with normal laboratory findings (p = 0.003). there were 26.72 % of patients with altered standard eeg, as well as 31.76 % of patients who underwent partially sleep-deprived eeg. 5 0% of patients who underwent mr angiography of the brain had altered findings. 36.67 % of patients who underwent vep testing had altered findings as well. there were 22.88 % of patients with altered ophthalmic findings and 46.43 % with altered orl findings. of all the patients who underwent an ecg, only one patient had altered findings, as well as pathologically changed continuous blood pressure monitoring. two out of five patients had altered lumbar puncture findings and two out of 11 patients who underwent an initial ct scan reported changes (table 5). table 5. test results n (%) p* laboratory data normal 20 (80) 0.003 altered 5 (20) eeg, standard normal 96 (73.28) < 0.001 altered 35 (26.72) eeg, partially sleep-deprived normal 58 (68.24) < 0.001 altered 27 (31.76) brain mri normal 42 (42.86) 0.669 altered 56 (57.14) mr angiography normal 3 (50) > 0.999 altered 3 (50) vep normal 19 (63.33) 0.144 altered 11 (36.67) ophthalmic findings normal 91 (77.12) < 0.001 altered 27 (22.88) orl examination normal 15 (53.57) 0.706 altered 13 (46.43) ecg normal 34 (71.14) < 0.001 altered 1 (2.86) pressure holter normal 3 (75) 0.317 altered 1 (25) lumbar puncture normal 3 (60) 0.655 altered 2 (40) head ct normal 9 (81.82) 0.035 altered 2 (18.18) *χ2 test statistically significant differences were found in the incidence of headache types and particular categories of psychological disorders (p = 0.024). significantly more disorders were reported in terms of emotional instability (15.15 %), cognitive deficits (15.15 %) and somatization (18.18 %) in functional headaches, while significantly higher levels of stress and similar conditions were seemedj 2020, vol 4, no. 2 neuroimaging in juvenile headaches 73 southeastern european medical journal, 2020; 4(2) observed with organic headaches (82 %) (table 6). table 6. psychological disorder and headache type functional organic p* n (%) emotional instability 5 (15.15) 4 (8) 0.024 cognitive deficits 5 (15.15) 3 (6) stress 17 (51.52) 41 (82) somatization 6 (18.18) 2 (4) *χ2 test extremely difficult psychological conditions in children required a psychiatric consultative examination. 10.79 % of patients underwent psychiatric examination as part of headache treatment (p < 0.001) (table 7). table 7. reasons according to consultative psychiatric examination n (%) p* extremely high depression level 1 (6.67) 0.395 in therapy because of suicidal thoughts and self-harm 2 (13.33) pronounced symptoms of somatization disorders and anxietyrelated disorders 1 (6.67) organic origin of headache excluded; in psychiatric therapy until further notice 1 (6.67) somatization headaches 2 (13.33) outpatient treatment started 5 (33.33) in therapy because of bullying at school 1 (6.67) identified ptsd symptoms due to loss of parent(s) 1 (6.67) multiple hospitalizations 1 (6.67) *χ2 test a significant difference was found in the proportion of final headache diagnoses. headaches as a consequence of psychological tension (26.62 %) account for the largest proportion (table 8). table 8. definitive headache diagnosis n (%) p head trauma 8 (5.76) < 0.001 sah 2 (1.44) tumor 0 (0) sinusitis 13 (9.35) acute infection 13 (9.35) inflammatory cns disease 1 (0.72) cessation of aet intake 2 (1.44) consequence of psychological tension 37 (26.62) papillary edema of the optic nerve 5 (3.5) *χ2 test seemedj 2020, vol 4, no. 2 neuroimaging in juvenile headaches 74 southeastern european medical journal, 2020; 4(2) discussion headache is the most common neurological symptom and one of the most common childhood pains (8). according to the literature, the estimated incidence of recurrent headaches in children is about 80 %, with 10 % of them experiencing headaches more than 5 days a month, which negatively affects both the child and family. the most common types of childhood headaches are migraines and tension-type headaches. studies have shown that learning disabilities have a high prevalence in children with primary headaches, especially migraines (8, 9). this study found that headache symptoms were more common in female subjects, as was the case in the study by wilcox et al. (10). adolescent girls are more prone to developing headaches than boys, and consequently, they are at a higher risk of migraine or other primary headaches in young adulthood (11). the number of newly diagnosed headaches in 2017 was 71 (51.0 %), and in 2018, it was 68 (48.92 %). the number of hospitalizations indicates that a significantly high number of patients was hospitalized on more than one occasion (58.7 %). despite the high prevalence of headaches in the pediatric population, the characteristics of adolescent headaches remain unclear. several studies have examined the age and genderrelated differences in migraines and a few reported differences between children and adults, as well as between younger and older children. the onset of migraines in younger children is often in the form of cyclic vomiting or abdominal migraines, and is not accompanied by a headache, while in older children, more complicated migraines are accompanied by focal neurological symptoms. due to the effect of hormones on the onset of migraines, migraines are 50% more likely to stop in males after adolescence (10, 12, 13). the transition from childhood to adolescence is a sensitive and critical period for neurodevelopment, especially in the context of neurological disorders such as migraine (10). the mean age of onset of puberty is about 10 in girls and about 12 in boys (2). in this study, headaches were more common in puberty, in children over the age of 12, in 69.78 % of cases. other studies confirm that migraine occurs in 3% to 10 % of children at puberty, increases with age and spontaneously withdraws after puberty in half of the children, but if it begins during adolescence, it is more likely to persist throughout adulthood (10, 12). in this study, children complained of 7 different headache localizations. the most common localization was frontal, with 40.29 % of the patients reporting it. somewhat less common were occipital 17.27 % and parietal 13.67 % headaches. an eight-year epidemiological study of unilateral headaches reported that unilateral primary headaches are more common in shortterm headaches (<4 hours), but long-lasting headaches, including migraine, tension-type headache and new daily persistent headache, can also occur with pain on one side (14). this study found that the most common side effects of headache were nausea and vomiting, which were reported by 61 subjects (44.85 %). antonaci et al. reported that recurrent primary headaches can have a significant impact on the quality of life of children and adolescents due to the unpredictability of attacks and the presence of concomitant symptoms such as nausea, vomiting, photoand/or phonophobia (5). the most important diagnostic tools in the correct diagnosis of headache type are meticulous medical history, clear medical history and clinical examination. one of the key components is the distinction between primary and secondary headaches (15). each patient admitted to the pediatric clinic with a headache, in addition to providing a detailed medical history and undergoing clinical and neurological examination, underwent complete neuroradiological assessment, ophthalmic and otorhinolaryngologic (orl) examination. in this study, 57.14 % of patients had altered magnetic resonance imaging (brain mri). the majority of the changes found were pineal gland cyst, vascular and gliotic lesions, and small demyelinating lesions. altered standard eeg findings were present in 26.72 % of patients, seemedj 2020, vol 4, no. 2 neuroimaging in juvenile headaches 75 southeastern european medical journal, 2020; 4(2) while 31.76 % of patients who underwent partially sleep-deprived eeg had altered findings as well. there were 6 (4.32 %) patients with clinical indications for additional neuroradiological processing (mr angiography), of which 3 (50 %) had pathological findings of the cerebral blood vessels. based on advanced neurological processing, the indication for vep was found in 30 (21.58 %) patients, 11 of whom (36.67 %) had pathological findings. there were 28 (20.14 %) patients with an indication for additional otorhinolaryngologic evaluation, 13 of whom (46.43 %) were diagnosed with sinusitis. invasive neurological treatment, which included lumbar puncture, was indicated in 5 (3.59 %) patients, and after the procedure, the findings were altered in 2 (40 %) patients, 1 of whom was diagnosed with an inflammatory disease of the central nervous system. 11 (7.91 %) patients had an urgent indication for computed tomography of the brain (ct), 2 of whom (18.18 %) presented with abnormalities (intracranial hemorrhage). all patients admitted to the pediatric clinic with anamnestic headache data were also subjected to psychological examination. findings thereof were extremely important in defining some of the psychological difficulties, as well as environmental factors and living conditions of each child. more recent studies, such as this one, have emphasized the importance of recognizing psychosocial components in a child with a headache for elucidating the etiology thereof, but also in order to be able to access therapeutic procedures based on adequate psychological analysis. events that greatly affect the incidence and type of headache include a range of family situations such as family conflicts and social problems, parental (un)employment, and economic hardships (16, 17). psychiatric disorders, especially depression, neurosis and anxiety, show significant comorbidity with migraine and are more common among people with chronic migraine compared with episodic migraine (18). in the group of children with functional headaches, more disorders were observed in terms of emotional instability (15.15%), cognitive deficits (15.15 %) and somatization (18.18 %), while in regard to organic headaches, there was a significantly higher level of stress and similar conditions (82 %). of all the definitive diagnoses of headaches, headache as a result of psychological tension is the most common diagnosis (26.62 %). conclusion this study aimed to research the incidence and frequency of headaches, to examine the correlation between comorbidity and the type of headache, and to examine whether there is a correlation between the psychological profiles of children at the time of onset of symptoms and the type of headache. this study confirmed that the highest proportion of definitive diagnoses of headaches were headaches that result from psychological tension. a smaller proportion are secondary headaches caused by inflammation of the central nervous system, sinusitis, trauma and subarachnoid hemorrhage. by preserving the mental health of children, we can prevent somatization, which is most common in puberty. acknowledgement. i would like to thank everyone who helped in the completion of this paper work. disclosure funding. no specific funding was received for this study. competing interests. none to declare. i references 1. šoštarić – kručaj z, i sur. glavobolja u dječjoj dobi. med. vjesn. 1988;2 0(1):31-33. 2. mardešić d. pedijatrija. 8 izd. zagreb: školska knjiga; 2016. 3. barišić n. pedijatrijska neurologija. zagreb: medicinska naklada; 2009. 4. straube a, heinen f, ebinger f and von kries r. headache in school children: prevalence seemedj 2020, vol 4, no. 2 neuroimaging in juvenile headaches 76 southeastern european medical journal, 2020; 4(2) and risk factors. dtsch arztbel int. 2013; 110(48):811–848. 5. antonaci f, voiticovschi-losob c, di stefano al, galli f, ozge a and balottin u. the evolution of headache from childhood to adulthood: a review od the literature. j headache pain. 2014 ;15(1):15. 6. wöber-bingöl c, wöber c, wagnerennsgraber c, zebenholzer k, vesely c, geldner j, karwautz a. ihs criteria and gender: a study on migraine and tension-type headache in children and adolescents. cephalagia. 1996; 16(2):107-12. 7. barišić n, prpić i, lehman i, grđan p, rešić b. smjernice hrvatskog društva za dječju neurologiju za dijagnostiku i liječenje glavobolja u djece. paediatrica croatia, 2012; 56:2. 8. genizi j, guidetti v, arruda ma. primary headaches and school performanc – is there a connection?. curr pain headache rep. 2017; 21(7):31. 9. abu-arafeh i, razak s, sivaraman b, graham c. prevalence of headache and migraine in children and adolescents: a systematic review of population-based studies. dev med child neurol. 2010; 52(12):1088-97. 10. wilcox sl, ludwick am, lebel a, borsook d. ageand sex-related differences in the presentation of paediatric migraine: a retrospective cohort study. cephalalgia. 2018; 38(6):1107-1118. 11. sillanpää m, saarinen mm. long term outcome of childhood onset headache: a prospective community study. cephalalgia. 2018; 38(6):1159-1166. 12. rothner ad. migraine variants in children. pediatr ann. 2018;47(2):e50-e54. 13. green a, kabbouche m, kacperski j, hershey a, o'brien h. managing migraine headaches in children and adolescents. expert rev clin pharmacol. 2016; 9(3):477-82. 14. leone m, cecchini ap, mea e, tullo v, bussone g. epidemiology of fixed unilateral headaches. cephalagia. 2008; 28 suppl 1:8-11. 15. gofshteyn js, stephenson dj. diagnosis and managment of childhood headache. current problems in pediatric and adolescent health care. 2016; 46(2):36-51. 16. russo a, bruno a, trojsi f, tessitore a, tedeschi g. lifestyle factors and migraine in childhood. curr oain headache rep. 2016; 20(2):9. 17. molarius a, tegelberg a, ohrvik j. socioeconomic factors, lifestyle, and headache disorders-a population-based study in sweden. headache. 2008; 48(10):1426-37. seng ek, buse dc, klepper je, mayson sj, grinberg as, grosberg bm et al. psychological factors associated with chronic migraine and severe migraine-related disability: an observational study in tertiary headache center. headache. 2017; 57(4):593-604. i author contribution. acquisition of data: serdarušić i, pušeljić s, tomac v, romić m administrative, technical or logistic support: serdarušić i, pušeljić s, tomac v, romić m analysis and interpretation of data: serdarušić i, pušeljić s, tomac v, romić m conception and design: serdarušić i, pušeljić s, tomac v, romić m critical revision of the article for important intellectual content: serdarušić i, pušeljić s, tomac v, romić m drafting of the article: serdarušić i, pušeljić s, tomac v, romić m final approval of the article: serdarušić i, pušeljić s, tomac v, romić m guarantor of the study: serdarušić i, pušeljić s, tomac v, romić m provision of study materials or patients: serdarušić i, pušeljić s, tomac v, romić m statistical expertise: serdarušić i, pušeljić s, tomac v, romić m seemedj 2020, vol 4, no. 2 percutaneous laser disc decompression and lumbar radicular pain 62 southeastern european medical journal, 2020; 4(2) short review article percutaneous laser disc decompression in the treatment of lumbar radicular pain 1 dino budrovac *1, ivan radoš 1, ozana katarina tot 1, ivana haršanji drenjančević 1, ivan omrčen 1 1 department of anaesthesiology, reanimatology and intensive care, university hospital centre osijek, croatia; faculty of medicine, university of osijek, croatia *corresponding author: dino budrovac, dino.budrovac@gmail.com received: apr 1, 2020; revised version accepted: sep 16, 2020; published: nov 12, 2020 keywords: herniated disc, laser, percutaneous diskectomy, radiculopathy abstract lumbar radicular pain is defined as pain in the lumbar spine with propagation to the lower extremities. it is a major public health, social and economic problem in the modern society, and is one of the most common reasons for visits to the doctor. lumbar radicular pain is often the reason for absenteeism and occupational disabilities. it is estimated that about 70-85% of the world’s population have experienced lumbar spine pain once in their lifetime. there are numerous modalities for the treatment of lumbar radicular pain, ranging from pharmacotherapy to surgery. in order to avoid systemic side effects of analgesics, anaesthesia and long-term and extensive surgery, minimally invasive procedures are increasingly used for treating lumbar radicular pain. percutaneous laser disc decompression (pldd) is one such procedure, first performed by dr choy and dr ascher in 1986. pldd is an outpatient surgery performed under local anaesthesia, its success rate is high and the complication rate is low. this method therefore certainly attracts the attention of clinicians dealing with this issue. (budrovac d, radoš i, tot ok, haršanji drenjančević i, omrčen i. percutaneous laser disc decompression in the treatment of lumbar radicular pain. seemedj 2020; 4(2); 62-68) seemedj 2020, vol 4, no. 2 percutaneous laser disc decompression and lumbar radicular pain 63 southeastern european medical journal, 2020; 4(2) introduction improper posture, sedentary lifestyle and frequent weightlifting are the most common causes of herniated intervertebral discs, which is the reason why young, working-age people suffer the most from such pain. it is estimated that 80% of people experience low back pain in their lifetime (1). lumbar pain with or without a radicular component most commonly occurs in adults, but it may also occur in the paediatric population (2). lumbar radicular pain is first treated conservatively, and if the pain persists, the gold standard is surgical microdiscectomy. in most patients who experience the first episode of lumbar radicular pain, it decreases to a level that does not impair daily functioning after 6 weeks (3). the increase in incidence of low back pain caused by herniated intervertebral disc increased the attention given to minimally invasive pain management (1). due to faster recovery than after surgical microdiscectomy, percutaneous laser disc decompression (pldd) has attracted much attention. the first pldd was performed in 1986 and the procedure was approved by the fda in 1991 (4). percutaneous laser disc decompression is a minimally invasive method in which the risk of damage to muscles, bones, ligaments and nerves is reduced. the aim of this study is to present pldd as one of the treatment modalities of lumbar pain caused by disc herniation. mechanism of pain onset lumbar radicular pain can be caused by compression of the herniated disc on the nerve, but it may also be caused by local inflammation. acute pressure on the nerve root causes numbness, paraesthesia, weakness and pain. disc pressure on the nerve root leads to disruption of nutritional supply to the nerve, increased permeability of blood vessels, impaired ionic balance and changes in the conduction of the nerve impulse. when there is no compression of the herniated disc on the nerve root, the cause of pain are substances from the nucleus pulposus, proteoglycans and a lowered ph level. when the integrity of the annulus fibrosus is impaired, a substance leaks into the epidural space, producing a proinflammatory effect. phospholipase a2 plays an important role in this process. much higher phospholipase a2 levels were observed in disc extrusion than in intact disc; this substance is involved in the synthesis of prostaglandin and leukotriene, leading to a local inflammatory response (5). mechanism of action of percutaneous laser disc decompression the mechanism of action of percutaneous laser disc decompression is based on the principle that the disc is viewed as a closed hydraulic system containing water, which is incompressible. the water content of the disc is about 50-89%, and decreases with age. laser energy warms the surrounding tissue. in this way, a small volume of nucleus pulposus water content is evaporated (figure 1). a small change in the volume of water results in a disproportionate decrease in pressure inside the disk (6). a decrease in pressure within the disc causes the hernia to withdraw and leads to a decrease in pressure on the root of the involved nerve. this reduces lumbar radicular pain caused by disc herniation. figure 1. the laser probe is inserted into the needle. evaporation of water from the nucleus pulposus (author’s work). seemedj 2020, vol 4, no. 2 percutaneous laser disc decompression and lumbar radicular pain 64 southeastern european medical journal, 2020; 4(2) anatomy of the intervertebral disc the intervertebral disc is a complex structure; it is the largest avascular structure in the body and it enables spinal mobility (7). it consists of the outer part, annulus fibrosus, and the inner part, nucleus pulposus (8). annulus fibrosus consists of 15-25 layers of crisscrossed fibres; the number of layers increases in the lumbar spine segment. annulus fibrosus consists of about 60% collagen and 20% proteoglycan, and nucleus pulposus consists of 65% proteoglycan and 20% collagen (9). annulus fibrosus is innervated by spinal nerve branches, while there is no innervation of the nucleus pulposus in an intact disc. the metabolism of the disc is mainly anaerobic and nutrients are diffused through the endplate. degeneration of the endplate thus results in insufficient nutritional supply of the intervertebral disc (10). reduced amounts of proteoglycans, collagen, water, and calcified endplate lead to disc damage. degenerative changes of the disc lead to a reduction in intervertebral distance and thus cause osteoarthritis of the facet joints. the incidence of degenerative disc changes increases with age, and if they occur in younger people, the cause is most often a genetic predisposition or injury. some of the factors that accelerate degenerative changes of the disc include smoking, atherosclerosis, frequent lifting of heavy loads, and a sedentary lifestyle. technique of performing percutaneous laser disc decompression pldd is performed in the operating room. during the procedure, the patient lies in a prone position. the skin at the intended puncture site is sterilized, prepared and protected from the surrounding area with sterile compresses. after the relevant intervertebral space (disc) is visualized, the optimal position is found by lateral and craniocaudal angulation. local anaesthetic infiltration is performed at the puncture site and an 18 g needle insertion is performed at the same place. the needle advances under fluoroscope control to be positioned in the middle of the disk, and the final position is confirmed by lateral projection and contrast application (figure 2). the laser probe is then inserted into the needle, supplying laser energy according to default parameters, which depend on the device and the protocol of each institution. laser energy leads to tissue heating and evaporation of a small volume of water within the nucleus pulposus (11, 12). figure 2. confirmation of proper needle position in intervertebral disc after contrast administration. intervertebral disc l5/s1 (author’s work). indications and contraindications an indication for pldd is symptomatic disc protrusion. the pain may be localized in the lumbar spine or it may radiate into one or both legs. discogenic pain is likewise an indication for pldd. discogenic pain is pain that is not of radicular origin, it occurs in the absence of spinal deformity, and it has no positive signs of nerve tension. the generators of discogenic pain are nociceptive fibres of the annulus fibrosus. the outer posterolateral portion of the annulus fibrosus is rich in sensory fibres. first degree spondylolisthesis, mild scoliosis, and osteoarthritis are not contraindications for pldd. prior surgery is also not a contraindication, unless it was a vertebral fusion or there are nerve root adhesions. pldd can also be performed if there is disc extrusion without sequestration. seemedj 2020, vol 4, no. 2 percutaneous laser disc decompression and lumbar radicular pain 65 southeastern european medical journal, 2020; 4(2) there are differing opinions, but the author of the method believes that pldd can also be used in spinal canal stenosis exacerbated by disc herniation (13). contraindications for pldd are acute pain that has not been treated conservatively, because 80-85% of acute pain disappears after resting, relaxation, non-steroidal anti-inflammatory drugs, and epidural administration of steroids and local anaesthetic. pldd is contraindicated in severe spondylolisthesis, severe scoliosis, metastatic cancer, vertebral compression fracture, compression of the nerve root with the bone, and the presence of free disc sequestration. advanced age is not a contraindication, but in the elderly, the amount of water in the disc is reduced, which is more pronounced in males. haemorrhagic diathesis, near-disc vertebral haemangioma, multiple sclerosis, demyelinating diseases, and systemic infections are also contraindications (14). postprocedural recommendations after pldd, the patient is recommended to rest and lie, as well as to abstain from sitting and walking. after the first day, it is recommended to limit sitting and walking to a maximum of 20 minutes. wearing a lumbar orthosis for two weeks in order to reduce mobility is likewise recommended. patients who do not do manual labour can return to work after 3 days, while those who do manual labour can return to work after 7-10 days. physical therapy can be performed after 1 week. antibiotic prophylaxis is recommended to prevent infection (14). complications of pldd possible complications of pldd include nerve root injury, cauda equina syndrome, bowel perforation, thermal injury along the needle path, and thermal necrosis of the endplate. paraspinal muscle spasm that causes patients discomfort is also described, and in more severe cases, a physical examination will show lateral curvature of the spine with a concavity to the side of spasm. muscle tension can be sensed by palpation. muscle spasm disappears after 3-4 days and does not affect the outcome of the treatment. benzodiazepine as a relaxant and local heat can be used to relieve the spasm. aseptic or infectious discitis are more serious complications. the most common cause of infectious discitis is staphylococcus aureus. symptoms usually occur 3-5 days after surgery and manifest as fever and pain of the affected disc and increased serum inflammatory parameters. if discitis is suspected, an emergency mri of the affected disc should be performed. this condition requires antibiotic treatment. the incidence is less than 1%. the diagnosis of aseptic discitis is made by ruling out septic discitis. signs and symptoms are the same as those for septic discitis, but there is no fever or elevated inflammatory parameters. the condition improves after several days of rest and administration of non-steroidal antiinflammatory drugs. inflammation of the sacroiliac joint is likewise possible. it usually occurs several days after an excellent response to pldd. the mechanism of pain onset is thought to be the cessation of lumbar radicular pain, which leads to the cessation of compensatory “locking” of the lumbosacral (ls) segment and sacroiliac (si) joint. this condition is treated by infiltration of the si joint with local anaesthetics and corticosteroids and non-steroidal antiinflammatory drugs (14). discussion patients who experience pain caused by herniation of the intervertebral disc and who do not respond to conservative treatment are candidates for surgery (15). surgical treatment is still the gold standard in the treatment of disc herniation, but classic surgery may impair spinal stability, so consideration should be given to minimally invasive treatments (16, 17). compared to discectomy/microdiscectomy, pldd has fewer postoperative complications and tissue injuries (18). a group of iranian researchers observed the impact of pldd on pain intensity as measured by the visual analogue scale (vas) and on disability as measured by the oswestry disability index (odi). the mean vas before the procedure was 6.70, and it was 2.60 after the seemedj 2020, vol 4, no. 2 percutaneous laser disc decompression and lumbar radicular pain 66 southeastern european medical journal, 2020; 4(2) procedure. the smallest pain reduction was observed in a 27-year-old man and was 43%, while the highest pain reduction was 71%, observed in a 45-year-old female. the mean odi before surgery was 31.03 and it was 20.60 after surgery, which is statistically significant. (p < 0.001) (11). pldd is a minimally invasive procedure and could in some cases be used as an alternative to surgical discectomy. the study compared the success rates and complications between pldd and microdiscectomy. there was no difference in efficiency between the two methods. the rate of reoperation in the surgical group was 21%, and it was 52% for pldd, which is higher than expected. although the rate of recurrence in the pldd group was relatively high, we can conclude that surgical discectomy was avoided in 48% of patients over a two-year period (19). a randomized controlled trial compared the efficacy of pldd and conventional microdiscectomy. the study was conducted on 115 patients who had lumbar radicular pain caused by an intervertebral disc hernia no larger than one third of the spinal canal. the rolandmorris disability questionnaire showed that, at 8 and 52 weeks, pldd is equivalent to conventional surgery. recovery was expected to occur faster with pldd. the rate of reoperation was significantly lower with conventional microdiscectomy (38% vs. 16%). at one-year follow-up, pldd proved to be equivalent to conventional microdiscectomy (20). a study comparing 500 microdiscectomies and 500 pldds was performed. in the microdiscectomy group, 428 patients (85.6%) had good or excellent results, as opposed to 419 in the pldd group (83.8%). the complication rate in the microdiscectomy group was 2.2% (11 patients), while there were no complications in the pldd group (21). the author of a different study, dr choy, presented the results of pldd over 17 years; this study included 1,275 patients and 2,400 performed pldds (neck, thoracic and lumbar segments). the success rate according to macnab criteria was 89%. the complication rate (infectious discitis) was 0.4%, and there were no cases of nerve root injury or spinal cord injury (22). although extrusion was previously considered as a contraindication, choy et al presented the results of a study that included 21 patients with disc extrusion without sequestration. eighteen patients experienced significant reductions in pain and, in some cases, a reversal of neurological deficits (23). a group of authors performed a systematic review and meta-analysis to compare the complication rates of different discectomy methods. the methods that were compared were open discectomy/microdiscectomy (od/md) with microendoscopic discectomy (med), percutaneous endoscopic lumbar discectomy (peld) and percutaneous laser disc decompression (pldd) and tubular discectomy. they found 17 randomized controlled trials and 20 cohort studies that met their criteria. metaanalysis of rcts showed that the overall complication rates for od/md, med, peld, pldd and tubular discectomies were 16.8%, 16.2%, 21.2%, 5.8%, 8.4% and 25.8%, respectively. reoperation rates were 8.4%, 4.7%, 6.7%, 23.2% and 11.7%, respectively. meta-analysis of cohort studies showed that overall complication rates were 7.6%, 6.2%, 9.1%, 3.5% and 11.6%, respectively. reoperation rates were 5.5%, 0.8%, 9.4%, 3.2% and 3.7%, respectively (24). patel and singh, in a retrospective study conducted on 65 patients treated with pldd, reported that the preprocedure vas score was 7.6/10 and at 2week, 6-week, 3-6 month intervals, it was 3.7/10, 4.3/10, and 4.1/10, respectively (25). proper patient selection for pldd treatment has shortterm and long-term benefits. conclusion percutaneous laser disc decompression is a minimally invasive procedure, with a low complication rate, high success rate, and rapid recovery. proper selection of patients with lumbar radicular pain caused by herniated intervertebral disc produces good results and may delay surgery or, in some cases, be an alternative to surgery. seemedj 2020, vol 4, no. 2 percutaneous laser disc decompression and lumbar radicular pain 67 southeastern european medical journal, 2020; 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4(2) analysis. eur spine j. 2014; 23(5):1021-43. doi: 10.1007/s00586-013-3161-2 17. benzakour t, igoumenou v, mavrogenis af, benzakour a. current concepts for lumbar disc herniation. int orthop. 2019; 43(4):841-51. doi: 10.1007/s00264-018-4247-6 18. abrishamkar s, kouchakzadeh m, mirhosseini a, tabesh h, rezvani m, moayednia a, ganjeifar b, mahabadi a, yousef e, kooshki am. comparison of open surgical discectomy versus plasma-laser nucleoplasty in patients with single lumbar disc herniation. j res med sci. 2015; 20:1133–1137. doi:10.4103/17351995.172979 19. brouwer pa, brand r, van den akker-van marle me, jacobs w ch, schenk b, van den berg-huijsmans aa, koes wb, arts ma, van buchem ma, peul wc. percutaneous laser disc decompression versus conventional microdiscectomy for patients with sciatica: twoyear results of a randomised controlled trial. interv neuroradiol. 2017; 23(3):313–24. doi: 10.1177/1591019917699981 20. brouwer pa, brand r, van den akker-van marle me, jacobs w ch, schenk b, van den berg-huijsmans aa, koes wb, van buchem ma, arts mp, peul wc. percutaneous laser disc decompression versus conventional microdiscectomy in sciatica: a randomized controlled trial. spine j. 2015; 15(5):857-65. doi: 10.1016/j.spinee.2015.01.020 21. tassi gp. comparison of results of 500 microdiscectomies and 500 percutaneous laser disc decompression procedures for lumbar disc herniation. photomed laser surg. 2006; 24(6):694–7. doi: 10.1089/pho.2006.24.694 22. choy ds. percutaneous laser disc decompression: a 17-year experience. photomed laser surg. 2004; 22(5):407-10. 23. choy ds. response of extruded intervertebral herniated discs to percutaneous laser disc decompression. j clin laser med surg. 2001;19(1):15-20. 24. chen x, chamoli u, castillo jv, ramakrishna v, diwan ad. complication rates of diferent discectomy techniques for symptomatic lumbar disc herniation: a systematic review and meta analysis. european spine journal. 2020; 29: 1752-70. doi:10.1007/s00586-020-06389-5 patel n, singh v. percutaneous lumbar laser discectomy: literature review and a retrospective analysis of 65 cases. photomed laser surg. 2018; 36:518–521. doi:10.1089/ pho.2018.4460 i i author contribution. acquisition of data: budrovac d, radoš i, tot ok, haršanji drenjančević i, omrčen i administrative, technical or logistic support: budrovac d, radoš i, tot ok, haršanji drenjančević i, omrčen i analysis and interpretation of data: budrovac d, radoš i, tot ok, haršanji drenjančević i, omrčen i conception and design: budrovac d, radoš i, tot ok, haršanji drenjančević i, omrčen i critical revision of the article for important intellectual content: budrovac d, radoš i, tot ok, haršanji drenjančević i, omrčen i drafting of the article: budrovac d, radoš i, tot ok, haršanji drenjančević i, omrčen i final approval of the article: budrovac d, radoš i, tot ok, haršanji drenjančević i, omrčen i seemedj 2020, vol 4, no. 2 stature estimation from the right external ear 12 southeastern european medical journal, 2020; 4(2) original article stature estimation from the right external ear of undergraduate students in south-east nigeria 1 sunday godwin obaje *1, sophia chinenye nwankwo 1, augustine ogugua egwu 1 1 department of anatomy, faculty of basic medical sciences, college of medical sciences, alex ekwueme federal university ndufu alike, ebonyi state, nigeria *corresponding author: obaje sunday godwin, obaje199@gmail.com received: may 13, 2020; revised version accepted: oct 27, 2020; published: nov 12, 2020 keywords: correlation analysis, external ear, regression models, sexual dimorphism abstract background: ethnicity, stature and gender influence the anthropometric characteristics of the right ear, thus creating variations which are helpful for sex identification and medico-legal purposes in forensic examinations. this study has produced anthropometric data on the right external ear among the ikwo people in southeastern nigeria. materials and methods: ear and lobular indices were obtained from the dimensions of the right external ear of 240 ikwo adults aged 18-35. also, three predictive models (equations) were produced in the study. results: descriptions of the right external ear for the study population were obtained. aside from ear indices, which were higher in females than in males, males were taller in stature than females (p<0.001). regarding correlation, right ear parameters in both sexes appeared positive and strongly correlated with stature. regression models strongly predicted stature based on external ear measurements. conclusion: medical applications (monitoring diseases, forensics, industrial design and apparel design) of this study cannot be overemphasized in this world of medical sciences, in particular when it comes to understand the human external ear in terms of hearing and communication. (obaje sg, nwankwo sc, egwu ao. stature estimation from the right external ear of undergraduate students in south-east nigeria. seemedj 2020; 4(2); 12-19) seemedj 2020, vol 4, no. 2 stature estimation from the right external ear 13 southeastern european medical journal, 2020; 4(2) introduction hearing, balance and communication are an integral part of human life (1). medical sciences, especially communication medicine, are raising medical standards to understand not only how the external ear works (hearing, balance and communication), but also to explain clinical disorders (2). by applying biochemical methods, forensic investigators collect information at a crime scene, such as fingerprints, hair strands, blood, even from notebooks, and trace the details of the person (3). in fact, the application of anthropometry to body structures has proved to be useful in forensics, genetics, agriculture, industry and neurosciences (4). anthropometry has been dealing with the measurement of physical features and functions of the human body, including linear dimensions, weight, volume and range of movement (5, 6). so, measuring the human ear is not an exception in this contemporary science. in another setting, scientists and anthropologists apply anthropometry to study human body measurements using parameters such as height, weight, triceps skinfold, subscapular skinfold, arm circumference, abdominal and calf circumference, knee height and elbow breadth (7-9). in terms of the anatomy, the ear is an advanced and very sensitive organ of the human body (10, 11). the ear's function is to transmit and transduce sound to the brain through the parts of the ear: the outer ear, the middle ear and the inner ear (11, 12). diseases and abnormalities of the ear are enormous. there are a number of different infections, diseases and even cancers that can affect the ear and ultimately lead to hearing loss (otosclerosis, otitis media and ménière’s disease) (13-15). stature estimations also include body height, usually estimated by applying anatomical and mathematical techniques using long bones (foot dimensions, hand and head measurements). it has been observed that in males, total height is sometimes greater and correlates more with stature than in females (11,16-18). the study using ear indices, lobular indices and stature of the ikwo people in southeastern nigeria will be useful in forensic science, ear health and clinical sciences. material and methods study location ebonyi state is located in southeastern nigeria and inhabited primarily by the igbo people, with the city of abakaliki as its capital. there are other major townships, including afikpo, onueke, ezzamgbo, edda, effium, aba omege, amasiri, unwana, echara ikwo, egu-ubia, ụbụrụ, onicha. the state covers a large area of 5,533 km². its latitude is 6°10'40.7"n and its longitude is 7°57'33.43"e. ebonyi has a tropical climate, with the summers much rainier than the winters. when it comes to agriculture, it is one of the most important states in nigeria, known for its popular brand of rice abakaliki and other food crops grown in large quantities, such as yam, cassava, maize, cocoyam, cowpea and groundnut. study design in the study, about 240 students (male = 120 and female = 120) from alex ekwueme federal university ndufu-alike in the 2018/2019 academic session of aged 18-35 years were recruited. the informed consent was obtained and signed. also, ethical approval (funai/re/120) was obtained prior to the study. a digital anthropometric method was applied to measure ear length and breadth, base of auricle, lobe length and breadth while stature measured using a stadiometer (figure 1 and figure 2). also, the ear and lobular indices, lobe attachments, and ear shape were calculated and from this, differentiating sex based on external ear variables was achieved. ear length was measured as the perpendicular distance between the tangents to the highest point on the helix and the lowest point on the lobule. ear index was measured as distance from the ear width/ear length ×100. lobular index was measured as the lobular width/lobular length ×100. seemedj 2020, vol 4, no. 2 stature estimation from the right external ear 14 southeastern european medical journal, 2020; 4(2) figure 1. the stadiometer used for measuring the stature of the participants from alex ekwueme federal university ndufu-alike, ebonyi state, nigeria stature was measured from the foot to the vertex of the head. all measurements were made by two research assistants using a scale to the nearest millimetre. figure 2. points used for anthropometric measurements of the right external ear among the subjects from alex ekwueme federal university ndufu-alike, ebonyi state, nigeria ****total ear height= l-h, ear width= a-p, lobular height= lt, lobular width (c-d). inclusion criteria all participants recruited for the study must belong to the same ethnic group (igbo). must be enrolled as students in alex ekwueme federal university ndufu-alike ikwo, ebonyi state, southern part of nigeria in the 2018/2019 academic session. also, subjects must belong to the age bracket of 18-35 years. exclusion criteria subjects that are non-igbo in the university were excluded from the study. statistical analysis the data was collected using excel spreadsheets (ms office, 2013). the statistical analysis of the data was performed using spss version 20 (ibm corp., armonk, ny). student’s ttest was performed after determining the normality of data (p<0.001). a pearson's product moment correlation coefficient was applied on the right ear for both sexes. also, the linear regression analysis was used to generate regression models to ascertain the level and strength of association between stature and right ear morphometry. results right ear indices (see table 1) were higher in females than in males (54.8±2.53 mm and 51.52±2.51 mm respectively), while males were taller in stature than females (161.42±7.81 mm and 160.21±7.01 mm respectively). lobular indices (99.45±8.62 mm and 99.43±8.61 mm) appeared to be the same in both males and females (p<0.001). all parameters appeared to be significant (p<0.001). seemedj 2020, vol 4, no. 2 stature estimation from the right external ear 15 southeastern european medical journal, 2020; 4(2) table 1. anthropometric measurements using the t-test to measure the right ear and stature among male and female adults of the ikwo people (mm) parameters (mm) males n = 120 females n = 120 p-values ei 51.52±2.51 54.81±2.53 0.001 li 99.45±8.62 99.43±8.61 0.001 s 161.42±7.81 160.21±7.53 0.001 *** ei: ear indices, li: lobular indices, s: stature the analysis (see table 2) showed a positive and stronger correlation in males than in females regarding the three parameters (p<0.001). in terms of correlation, ear indices were positive and stronger in males than in females (r = 0.92 and r = 0.82 respectively), while the value of lobular indices was r = 0.90 and 0.86 respectively. furthermore, correlation between the ear variables with stature were positive and strong in both males and females (r = 0.89 and r = 0.80 respectively). in the regression analysis (see table 3 and 4), r2 adjusted values were 0.94, 0.91 and 0.85 for ear indices, lobular indices and stature for males, and 0.90, 0.89, and 0.65 for females respectively. in this case, the study appeared strongly predictive regarding the three ear measurements with stature for both sexes and statically significant (p<0.001). table 2. correlational analysis of the right ear and stature among male and female adults of the ikwo people parameters male stature female stature r p-value r p-value ei 0.92 0.001 0.82 0.001 li 0.90 0.001 0.86 0.001 s 0.89 0.001 0.80 0.001 ***el: ear length, ew; ear width, ll: lobular length, lw: lobular width, ei: ear indices, li: lobular indices, s: stature predictive equations for ear indices, lobular indices and stature were s = 89.14+(1.61×ei), 88.71+(1.71×li) and 74.45+(1.68×s) for males, and s = 88.12+(1.60×ei), 86.27+(1.68×li) and 74.45+(1.66×s) for females respectively. discussion scientists studying human ears are adding new dimensions to future discussions about the external ear in humans (1, 2). our study showed that males were taller in stature than females. this also corroborated the sexual dimorphic features studied in anthropology. moreover, ear indices appeared to be higher in males than in females, while the lobular indices were the same in both sexes. the human ear gives a clue to estimating stature at the time of criminal investigations and legal discussions (2, 5). in northern nigeria, a study of the hausa ethnic group aged 16-35 used the digital and face-arth software and observed sexual dimorphism in ear seemedj 2020, vol 4, no. 2 stature estimation from the right external ear 16 southeastern european medical journal, 2020; 4(2) analyses (12, 17). also, a comparative study of the external ear involving medical students showed that all ear and lobular indices were higher in males than in females (13, 18, 19). the morphometric variables of the right female ear were somewhat inconsistent with our study, as they were higher compared to the right ear in males, although the difference was not significant (20, 21). the reason for the differences observed in the study can be attributed to the influence of ethnicity, genetics, climate and nutrition (22-24). our study follows the application of krogman's principle to determine regression formulae used to estimate stature from bones, fragments of bones or measurements of body parts (2), and it was reported that regression formulae presents a better estimation and association between ear indices and stature. our study (table 3, 4) produced three prediction models (ear indices, lobular indices and stature). table 3. regression equations for predictions of stature from the right ear among male adults of the ikwo people model male r2 p-value r-equation ei 0.94 0.001 s = 89.14+(1.61×ei) li 0.91 0.001 s = 88.71+(1.71×li) s 0.85 0.001 s = 74.45+(1.68×s) *** ei: ear indices, li: lobular indices, s: stature table 4. regression equations for predictions of stature from the right ear among female adults of the ikwo people model female r2 p-value r-equation ei 0.90 0.001 s = 88.12+(1.60×ei) li 0.89 0.001 s = 86.27+(1.68×li) s 0.65 0.001 s = 74.45+(1.66×s) *** ei: ear indices, li: lobular indices, s: stature all the regression-adjusted values (r2) for ear indices, lobular indices and stature were positive and higher in males than in females. all the anthropometric parameters were significant (p<0.001). some studies (25-28) were consistent with our study that predicted stature from both ears in a simple linear regression analysis. furthermore, some studies implicated the evolutionary changes to have influenced ear measurements (29-30). again, interracial marriages have become the norm in many cultures and have been producing genetically similar population (31). apart from ear dimensions, some studies deal with other human body parts to estimate stature using regression models (32). the similarity in lobular indices in our subjects (table 1-2) may result from the fact that they all belong to the same generation, with great grandparents who carried the same genetic interpretation. regarding sexual dimorphism, there reference to the correlation and estimation of stature from other body parts, for instance in a study of estimation of stature from length of fingers, estimation of stature from index and ring finger seemedj 2020, vol 4, no. 2 stature estimation from the right external ear 17 southeastern european medical journal, 2020; 4(2) length and estimation of stature from hand and phalange length (32-33). also, studies on the estimation of stature from craniofacial measurements, sex differences in morphometry of north indian acetabula (34-35). in particular, in some cases, it has been pointed out that the reason behind the association between stature and anatomical parts could be traceable to protein intake in early childhood with body composition, insulin-like growth factor in midchildhood and early adolescence, environmental and genetic factors (21, 26). importantly, human beings are considered to be bilaterally symmetrical in both thumb and fingers of both hands, irrespective of sex or handedness, allowing for an estimation of stature of an individual, which plays an important role for individuality (33, 35). to further support our study, one research discussed the right and left ears as mostly asymmetrical with respect to ear length, ear breadth and base of auricle, which were larger in males than in females, while the lobe length and breadth were larger in females as compared with males (chi square test, p ⩽ 0.001), concluding that the ear parameters gave a moderate to good sex identification accuracy (36). this study could be useful for human embryology and monitoring gestational age, for industrial designs and forensic education. conclusion ear and lobular indices appear to be correlated with stature. using the three regression models, a correlation between stature and ear measurement can be predicted, meaning that ear length can be used to identify individual stature. this study is helpful for communication medicine, forensic study and basic medical science. acknowledgement. our study team wish to thank prof uche nwachi edward, former provost of the college of medicine, for his administrative support and scientific advice. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. 1. grenness c, hickson l, laplantelévesque a, meyer c, davidson b. communication patterns in audiologic rehabilitation history-taking: audiologists, patients, and their companions. ear and hearing. 2015; 36(2);191-204. 2. van de water tr. a regenerative medicine approach to the treatment of hearing, balance, and olfactory disorders: what is in the future for otolaryngology? the anatomical record: 2020; 303(3):385-389. 3. ishikawa t, ishikawa t. forensic medicine and human cell research. springer singapore 2019. 4. saitou n, omoto k. time and place of human origins from mt dna data. nature. 1987; 327(6120):288-288. 5. kumar v, prakash a, pandya p, raina a. telomere: a forensic approach for age estimation. anil aggrawal's internet j forensic med toxicol. 2019; 20(1). 6. li z, miao f, yang z, wang h. an anthropometric study for the anthropomorphic design of tomato-harvesting robots. computers and electronics in agriculture. 2019; 163, 104881. 7. zakaria n, gupta d. (eds.). anthropometry, apparel sizing and design. woodhead publishing 2019. 8. bragança s, castellucci i, costa e, arezes p, carvalho m. anthropometric data for wheelchair users: a systematic literature review. seemedj 2020, vol 4, no. 2 stature estimation from the right external ear 18 southeastern european medical journal, 2020; 4(2) international journal of occupational safety and ergonomics. 2020; 26(1):149-172. 9. tang, h. k., bowe, s. j., nguyen, t. h., & dibley, m. j. triceps and subscapular skinfold thickness percentiles of a school-based sample of adolescents in ho chi minh city, vietnam. european journal of clinical nutrition. 2020; 1-5. 10. smaruj m, orkwiszewska a, adam m, jeżyk d, kostrzewa m, laskowski r. changes in anthropometric traits and body composition over a four‐year period in elite female judoka athletes. journal of human kinetics. 2019; 70(1):145-155. 11. meenderink sw, shera, ca, valero md, liberman mc, abdala c. morphological immaturity of the neonatal organ of corti and associated structures in humans. j association res otolaryngol. 2019; 20(5):461-474. 12. heine, p. a. 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(pp. 34-42). 15. cameriere r, deangelis d, ferrante l. ear identification: a pilot study. j forensic sci. 2011; 56(4):1010-1014. 16. das s, bansal m. variation of gene expression in plants is influenced by gene architecture and structural properties of promoters. plos one. 2019;14(3):e0212678 17. dhanda v, badhan j s, garg rk. studies on the development of latent ear prints and their significance in personal identification. problems of forensic sciences. 2011; 88:285-295. 18. gaya aa. yahaya ai. sub-saharan human morphological variations in the external ear (pinna): a potential tool for human identification. ec clinical and experimental anatomy. 2019; 2:175-184. 19. hwang ic, park ym, kang wc, moon j. association between height and lipid profile among korean men: results from the 10-year korea national health and nutrition examination survey. europ j preventive cardiol. 2019; 2047487319877055. 20. abdelaleem s, abdelbaky ff. estimation of stature in upper egypt population from external ear morphometry. int j forensic sci pathol. 2016; 4(10):276-284. 21. karavuş m, karaketir şg, hıdıroğlu s, lüleci ne, çetinkaya cy. hoşgör eb, karaketir s, karavuş a. a cross-sectional study evaluating childhood autism awareness of residents otorhinolaryngology department in i̇stanbul. istanbul medical journal = istanbul tip dergisi, 2019; 20(5):450. 22. spiller mw, gile kj, handcock ms, mar cm, wejnert c. evaluating variance estimators for respondent-driven sampling. j survey statistics methodol. 2018; 6(1):23-45. 23. laxman ka. study of determination of stature in hyderabad population from external ear morphometry. medico-legal update. 2019; 19(1):164-168. 24. merrill rm, fowers r. to what extent does sex, age and bmi impact medical and pharmacy costs? a retrospective cohort study involving employees in a large school district in the usa. bmj open. 2019; 9(5):e024078. 25. meijerman l, van der lugt c, maat gj. cross‐sectional anthropometric study of the external ear. j forensic sci. 2007; 52(2):286-293. 26. murgod v, angadi p, hallikerimath s, kale a. anthropometric study of the external ear and its applicability in sex identification: assessed in an indian sample. aust j forensic sci, 2013; 45(4):431-444. 27. rowsey dm, heaney lr, jansa sa. tempo and mode of mandibular shape and size evolution reveal mixed support for incumbency seemedj 2020, vol 4, no. 2 stature estimation from the right external ear 19 southeastern european medical journal, 2020; 4(2) effects in two clades of island‐endemic rodents (muridae: murinae). evolution 2019; 73(7):14111427. 28. shotwell m. the misuse of genetics: the dihybrid cross and the threat of “race crossing”. the american biology teacher. 2019; 81(1):3-10. 29. annie j, balaraj b, kumar p. a study of estimation of stature from length of fingers in mysore. indian j forensic med toxicol. 2010; 4(2):12–13. 30. bardale rv, dahodwala tm, sonar vd. estimation of stature from index and ring finger length. j indian academy forensic med. 2013; 35(4):353–357. 31. holland td. estimation of adult stature from fragmentary tibias. j forensic sci. 1998; 37:1223–1229. 32. neel jv, ward rh. the genetic structure of a tribal population, the yanomama indians. vi. analysis by f-statistics (including a comparison with the makiritare and xavante. genetics. 1972; 72(4):639-666. 33. özaslan a, i̇şcan my, özaslan i, tuğcu h, koç s. estimation of stature from body parts. forensic sci internat. 2003; 132(1):40-45. 34. danborno b, adebisi ss, adelaiye ab, ojo sa. estimation of height and weight from the lengths of second and fourth digits in nigerians. internet j forensic sci. 2009; 3(2):1-6. 35. matheswaran g, vallabhajosyula r. digit length displays a significant fraction in stature estimation: a study from coastal region of south india. int j anat res. 2014; 2(2):336-39. shaikh mmm, garg sm, shah ka. estimation of stature from craniofacial measurements. int j med toxicol legal med. 2019; 22(3and4): 118-122. i i author contribution: acquisition of data: obaje sg, nwankwo sc, egwu ao administrative, technical or logistic support: obaje sg, nwankwo sc, egwu ao conception and design: obaje sg, nwankwo sc, egwu ao critical revision of the article for important obaje sg, nwankwo sc, egwu ao final approval of the article: obaje sg, nwankwo sc, egwu ao seemedj 2020, vol 4, no. 2 liver peroxidation and limb ischemia-reperfusion injury 1 southeastern european medical journal, 2020; 4(2) original article experimental liver peroxidation against the background of limb ischemia reperfusion injury – is there a pathogenic difference between its modifications? 1 nataliya v. volotovska*1 1 department of physiology, bioethics and biosafety, i. ya. horbachevsky ternopil national medical university, ternopil, ukraine *corresponding author: nataliya volotovska, volotovskanv@tdmu.edu.ua received: feb 29, 2020; revised version accepted: jul 30, 2020; published: nov 12, 2020 keywords: ischemia-reperfusion syndrome, liver, blood loss, tourniquet, lipid peroxidation abstract introduction: use of the haemostatic tourniquet is an important step in providing first aid in open bleeding injuries. tourniquet pressure on the extremity triggers local lipid peroxidation. however, the systemic influence of tourniquet thereof has not been fully studied. aim: this study aimed to evaluate the changes that occur against the background of ischemiareperfusion syndrome (irs) and modifications of trauma in the main gland for detoxification – the liver. material and methods: in order to estimate the liver condition under the effects of a haemostatic tourniquet, animals were divided into five experimental groups, where modifications of hypoxia were performed as a result of bleeding, irs and trauma due to mechanical fracture of the thigh. results: biochemical study of the liver has shown that each type of such interventions caused the activation of lipid peroxidation in this organ. the highest increase of the malonic dialdehyde rate was observed in response to haemostatic tourniquet combined with blood loss. additionally, its content was higher in the group combining mechanical trauma and the tourniquet compared to isolated trauma. conclusion: all types of interventions caused hypoxia – as a result of isolated bleeding and cessation of blood flow due to the tourniquet. however, the release of overconcentration of toxic derivatives of rhabdomyolysis, which entered the blood stream after limb release, activated the pathological mechanisms of irs, which included intensified lipid peroxidation in the liver. (volotovska n. experimental liver peroxidation against the background of limb ischemia reperfusion injury – is there a pathogenic difference between its modifications? seemedj 2020; 4(2); 1-11) seemedj 2020, vol 4, no. 2 liver peroxidation and limb ischemia-reperfusion injury 2 southeastern european medical journal, 2020; 4(2) introduction nowadays, as in the past, the use of the haemostatic tourniquet is an important step in providing first aid [1, 2]. however, a careful study of the mechanisms of ischemia-reperfusion syndrome due to the use of haemostatic bandage is required [3]. the latter was proved to be an important cause of oxidative stress [4, 5], which promotes lipid peroxidation in the limb region under tourniquet pressure, and affects metabolism and protective reactions in the entire organism [6, 7]. the essence of ischemia-reperfusion syndrome (irs) is that after the release of the tourniquet, a large amount of toxins enters systemic blood circulation [8, 9], which, in turn, causes an increase in functional liver activity [10-13]. however, against the background of hypemic hypoxia, full liver repair is impossible. at the moment, there is not enough data that could comprehensively explain the correlation between the irs and the liver, especially lipid peroxidation (lpo) in the liver in the condition of irs material and methods the experiments were performed on 260 white non-linear male rats 5-5.5 months old. the animals were removed from the experiment at the 1st hour after intervention, and on the 1st, 3rd, 7th and 14th day after trauma on the basis of thiopental-sodium anaesthesia (40 mg/kg of body weight intraperitoneal) by total bleeding from the heart. such number was based on the need to obtain statistically significant data in each group, as well as at each time point – in order to see the stages of development of posttraumatic disease, in particular lipid peroxidation activity, caused by haemostatic tourniquet. the high number of experimental animas used is also due to higher mortality of rats in eg3 and eg5 groups (explained bellow) compared to other experimental groups animals were divided into 5 groups (n=10 animals per group): control group (cg), where rats were administered only thiopental-sodium anaesthesia (40 mg/kg of body weight intraperitoneally); eg1 (rubber tourniquet was applied to the upper third of the thigh for 2 hours, reperfusion lasted 1 hour (isolated irs); eg2 (simulated blood loss in the amount of 40% of the volume of circulating blood from the femoral vein); eg3 (tourniquet on thigh was combined with 40% blood loss from the femoral vein on the other lower limb), eg4 (mechanical trauma that caused fracture of femur), eg5 (tourniquet on thigh was combined with fracture of femur of the other lower limb). given the onset of severe pain in eg4 and eg5, an injection of a 2%-solution of lidocaine was administered for 7 days in the posttraumatic period. animals in other groups were administered analgesics twice – on the day of intervention and on the following day. the experiments were performed in the vivarium of i. horbachevsky tnmu in the morning. the special room had a stable temperature (18-22 ºc), relative humidity (4060%) and illumination of 250 lux. all experimental stages of work were performed in accordance with the european convention for the protection of vertebrate animals used for experimental and other scientific purposes (strasbourg, 1986), the resolution of the first national congress on bioethics (kyiv, 2001) and the order of the ministry of health of the ukraine no. 690 of 23 september 2009. the activity of active thiobarbituric acid derivatives in 10% of liver homogenate samples was determined using a method based on the ability of secondary products of lipid peroxidation (lpo), especially malonic dialdehyde, during a reaction with thiobarbituric acid at high temperatures and in acidic ph, to form a coloured complex with optic density that can be registered by spectrophotometry on waves of 532 nm [14]. statistical analysis statistical analysis of the obtained data was performed using excel (microsoft, usa). the seemedj 2020, vol 4, no. 2 liver peroxidation and limb ischemia-reperfusion injury 3 southeastern european medical journal, 2020; 4(2) statistical significance of the differences between independent indices was determined using student’s t-test at a normal distribution and by non-parametric methods in other cases. the correlation coefficient was significant at p < 0.05. results as can be seen in the data in figures 1 and 2 against the background of ischemia modelling, the content of tba-active products increases significantly. thus, in isolated ischemia-reperfusion injury (eg1) after the 1st hour, on the 1st, 3rd and 7th day, there was an increase of the index by 76.7% (p < 0.05), 2.8 times (p < 0.05), 2.5 times (p < 0.05) and 51.6% (p < 0.05), respectively, compared to the control. on the 14th day, the indicator did not return to baseline, exceeding the control by 25.9% (p > 0.05). against the background of isolated blood loss (eg3), the content of tba-active derivatives of lipid peroxidation after the 1st hour was 2 times higher than said index in cg (p < 0.05), on the 1st day after intervention 4 times (p < 0.05), on the 3rd day 3.5 times (p < 0.05), on the 7th day 2.8 times (p < 0.05) and on the 14th day 2.4 times higher (p < 0.05). as for eg3 – tourniquet on thigh was combined with 40% blood loss from the femoral vein on the other lower limb – that group included the most obvious increase in peroxidative activity compared to other groups: after the 1st hour, the index exceeded the control 2.4 times (p < 0.05), on the 1st, 3rd, 7th and 14th day, it was 3.8 times (p < 0.05), 4.9 times (p < 0.05), 3.6 times (p < 0.05) and 2.7 times higher (p < 0.05), respectively. figure 1 – dynamics of tba-active derivatives of lipid peroxidation in the liver (in % compared to the control level) after ischemia-reperfusion of limb and blood loss notes: statistical differences between the 1st day, 3rd day, 7th day and 14th day in comparison with the 1st hour, 1st day, 3rd day and 7th day, respectively are significant, p < 0.05) in the 1st group – * statistical significance compared to previous day in the 2nd group – ** statistical significance compared to previous day in the 3rd group – *** statistical significance compared to previous day seemedj 2020, vol 4, no. 2 liver peroxidation and limb ischemia-reperfusion injury 4 southeastern european medical journal, 2020; 4(2) comparison of the following groups with the effect of isolated ischemia-reperfusion of the limb on the lpo in liver tissue confirmed its pathogenic effect. thus, against the background of isolated mechanical trauma (mt) of the thigh (eg4) in the 1st hour after the intervention, the activity of the lpo increased by 32.8% (p > 0.05). significant increase was observed on the 1st, 3rd and 7th day after trauma, when, compared to the control, the index was higher by 65.6% (p > 0.05), 86.2% (p < 0.05) and 54.5% (p < 0.05), respectively. on the 14th day, the index did not differ significantly from the control. at the same time, its activity against the background of mt combined with the tourniquet (eg5) was higher. thus, after the 1st hour, the index was higher than in cg by 66.1% (p < 0.05). on the 1st and 3rd day, it remained at the same level – it was higher compared with the control by 2.8 times (p < 0.05) and 2.9 times (p < 0.05); on the 7th and 14th day, it exceeded it by 86.2% and 31.8%, respectively. figure 2 – dynamics of tba-active derivatives of lipid peroxidation in the liver after ir of limb and mechanical trauma notes: data are presented in % compared to the control level. statistical differences between the 1st day, 3rd day, 7th day and 14th day in comparison with the 1st hour, 1st day, 3rd day and 7th day, respectively, are significant, p < 0.05) in the 3rd group – *statistical significance compared to previous day in the 4th group – **statistical significance compared to previous day in the 5th group – ***statistical significance compared to previous day seemedj 2020, vol 4, no. 2 liver peroxidation and limb ischemia-reperfusion injury 5 southeastern european medical journal, 2020; 4(2) the dynamics of changes in the studied index had specific patterns caused by both ischemia and additional effects of mechanical trauma. with varying intensity, depending on the combination of pathogenic effects, the activity of lpo increased by the 3rd day, but decreased by the 14th day, although it did not reach the normal level of cg. thus, there was a significant increase of the index in eg1 – on the 1st day after the intervention, it remained increased by 58.8% (p < 0.05), compared with the activation of the pol in the 1st hour, after which a decrease was noted. at the same time, on the 3rd day it exceeded the activity in the 1st hour by 42.2% (p < 0.05). on the 7th and 14th day, the index was lower than the most acute period of the 1st day by 46% (p < 0.05) and by 1% (p < 0.05), respectively, and significantly lower than the 1st hour index. in eg2, the dynamics of activity of lpo derivatives was similar, with the highest score on the 1st day, when the index exceeded the data of the 1st hour by 96.1% (p < 0.05), after which it decreased markedly. thus, activity on the 7th day was lower by 30.5% (p < 0.05) and by 20.6% (p < 0.05) than indices of the 1st and 3rd day, respectively. with regard to the value of the index in eg3 on the 1st day, compared to the 1st hour, the index increased significantly by 58.8% (p < 0.05), on the 3rd day it increased by 27.6% (p < 0.05) compared to the 1st day, and 2 times compared to the 1st hour. the 7th day index was 27% lower than the 3rd day index (p < 0.05), and the 14th day index was 23% lower (p < 0.05) compared to the 7th day period. a significant increase in lpo activity was recorded on the 1st day in eg4, when the index exceeded the data obtained in the 1st hour after the intervention by 25.7% (p < 0.05). the highest activity score was on the 3rd day, when the index exceeded the data of the 1st hour by 40.4% (p < 0.05), after which it gradually decreased, becoming lower than the analogous index after the 1st hour, the 1st, 3rd and 7th day by 20.2% (p < 0.05), 36.5% (p < 0.05), 43.1% (p < 0.05) and 36.5% (p < 0.05), respectively. lpo activity in eg5, in contrast to eg1 and eg4, in which the 1st day was a critical period, continued rising on the 3rd day, when it exceeded the level of the 1st hour and 1st day by 70.4% (p < 0.05) and 72.8% (p < 0.05), respectively. after that, lpo activity decreased sharply – on the 7th day it decreased by 35.1% (p < 0.05) compared to the 3rd day, and on the 14th day it decreased by 29.3% (p < 0.05), compared to the 7th day. as can be seen in table 1, by comparing the values of the studied index in groups with different severity of ischemia, it was found that in the 1st hour, in eg3 the index was higher than in eg2 and eg1 by 26.5% (p < 0.05) and by 15.3% (p < 0.05), respectively. in addition, the index in eg1 was significantly higher than in eg4 – by 25% (p < 0.05). the results in eg5 exceeded eg4 by 20.2% (p < 0.05). thus, dynamics of lipid peroxidation were as follows: on the 1st day, the estimated index was lower than in eg2 and eg3 by 29.8% (p < 0.05) and by 26.5% (p < 0.05), respectively. also, in eg1, which was almost identical to eg5, it was higher than in eg4 (isolated trauma) by 40.6% (p < 0.05). on the 3rd day, there was a further increase in the activity of lpo in eg3 and eg5 and a decrease in the groups eg1, eg2 and eg4. index of eg3 was higher by 48.5% (p < 0.05) and by 28.3% (p < 0.05) than in eg1 and eg2, respectively. a comparison between the indices in eg1, eg4 and eg5 found that the index of eg1 was slightly lower than in eg5 – by 12.4% (p < 0.05) and higher than in eg4 by 26% (p < 0,05). on the 7th day, a sharp decrease of index dynamics was observed in all studied groups. the activity of lpo in eg3 remained higher than in eg2 and eg1 by 22% (p < 0.05) and by 57.4% (p < 0, 05), respectively, while the score in eg5 remained higher than in eg1 by 17% (p < 0.05). on the 14th day, the index in eg3 exceeded the indices in eg2 and eg1 by 11.9% (p < 0.05) and 2.2 times (p < 0.05), respectively. at the same time, the index of eg5 on the 14th day remained statistically significantly higher than in group 4, by 19.7% (p < 0.05), which confirms the seemedj 2020, vol 4, no. 2 liver peroxidation and limb ischemia-reperfusion injury 6 southeastern european medical journal, 2020; 4(2) attachment of tangential pathogenic effects caused by ischemia-reperfusion syndrome. table 1. content of tba-active derivatives of lipid peroxidation in 10% of rat liver homogenates based on variants of ischemia-1) group reperfusion period 1st hour 1st day 3rd day 7th day 14th day control = 1.89 (n = 10) group 1 isolated ischemiareperfusion 3.34 (n = 10) 5.22* (n = 10) 4.75* (n = 10) 2.87* (n = 10) 2.38 (n = 10) group 2 blood loss 3.85* (n = 7) 7.55* (n = 7) 6.61* (n = 6) 5.25* (n = 7) 4.54* (n = 7) group 3 ischemia-reperfusion + blood loss 4.55* (n = 6) 7.22* (n = 6) 9.22* (n = 6) 6.73* (n = 6) 5.15* (n = 5) р1-3  0.05  0.05  0.05  0.05  0.05 р2-3  0.05  0.05  0.05  0.05  0.05 group 4 trauma 2.51 (n = 10) 3.13* (n = 10) 3.52* (n = 10) 2.92* (n = 10) 2.00 (n = 10) group 5 ischemia-reperfusion + trauma 3.14* (n = 9) 5.35* (n = 9) 5.43* (n = 8) 3.52* (n = 9) 2.49* (n = 9) р1-5  0.05  0.05  0.05  0.05  0.05 р4-5  0.05  0.05  0.05  0.05  0.05 notes: 1.* – 2. р1-3 – the probability of differences in relation to experimental groups 1 and 3; 3. р2-3 – the probability of differences in relation to experimental groups 2 and 3; 4. р1-5 – the probability of differences in relation to experimental groups 1 and 5; 5. р4-5 – the probability of differences in relation to experimental groups 4 and 5 discussion this experimental study was dedicated to identifying the effects of ischemia-reperfusion syndrome of the limb on indices of lipid peroxidation in the liver tissue. it was estimated that even with isolated use of a tourniquet, as well as with isolated blood loss or injury, peroxidation processes in the liver were intensified. however, the addition of iri statistically significantly complicated the course of injuries, indicating a side effect of the use of the tourniquet. knowledge of the periodization of traumatic disease against the background of the studied pathology is important for the development of sanogenic effects in order to minimize this pathogenic factor. according to the latest research, haemostatic tourniquet causes not only temporary ischemia, but is accompanied by complications such as: ischemia and nerve damage in the pressure zone of the tourniquet, limb oedema, skin pallor, muscle weakness without paralysis and pain [9, 15]. in our experiment, we paid attention to the processes of peroxidation in the liver. although the tourniquet is an effective tool for rapid cessation of critical bleeding, its use for 2-3 hours can trigger irreversible processes of tissue death due to absence of blood supply and, in severe cases, can lead to limb amputation [1618]. correlation analysis showed that in the development of ischemia-reperfusion syndrome, caused by the application of a haemostatic tourniquet, obviously insufficient liver function could play a great role [19]. formation of the active hepatic response is a predictable reaction, which is part of the syndrome of multiple organ failure. contribution of the irs as the reason for such pathological effects to the organism was already seemedj 2020, vol 4, no. 2 liver peroxidation and limb ischemia-reperfusion injury 7 southeastern european medical journal, 2020; 4(2) demonstrated [20 25]. in addition to that, increased lipid peroxidation is manifested in certain liver dysfunction, especially bile formation [26]. present results confirmed the increase of liver lipid peroxidation in the presence of different conditions that cause ischemia: tourniquet and bleeding, combination of blood loss with haemostatic plait during the period, which is considered to be safe, but unfortunately further increases the degeneration of cell membranes. thus, the results of different scientists examined the concentration of glycolytic metabolites and the content of high energy phosphates in skeletal muscles, which were analysed at various times of tourniquet ischemia during operation in bloodless field. against the background of moderate growth of phosphocreatine in the ischemic limb, a pronounced increase of lactate (4-5 times) was observed. it is obvious that the reason for a moderate increase of glucose and glucose-6phosphate was the activation of glycogenolysis. since ischemia lasted from 30 to 90 minutes in these cases, the changes in indices were detected after 5 minutes [27]. present study did not directly show a sharp increase of lpo in the tissue possibly due to depletion of the antioxidant system in the liver. similar results, extraordinary increase (14 times) of lactate, were obtained by another group [28]. they likewise determined a decrease of skin temperature from 35.9 to 33.5 degrees, which quickly dropped to that level in the first 15 minutes, after which it remained stable at this level [28]. summing up, the main causes for activation of pathogenesis branches of ischemia-reperfusion injury became known as the following: rhabdomyolysis and hypoxia. the former resulted in hyperejection of toxins into the bloodstream after release of limb from tourniquet. its basic pathogenesis – muscle oedema with subsequent development of hypovolemia, haemoconcentration and massive release of myoglobin, potassium and biologically active substances that could trigger multiorgan failure from compressed tissue [29]. increased concentration of myoglobin in renal tubules in the conditions of acidic ph promotes intratubular obstruction and kidney dysfunction [30-32]. another factor is hypoxia, which is known to be the cause that stimulates lipid peroxidation [33] – and in our experiment it was activated by two causes – bleeding and ischemia. the latter provided not only the lack of oxygen, but also local compression with subsequent rhabdomyolysis. thus, a pathological circle was formed. in turn, liver function was recognized in this study as a protective reaction. the most severe period of lpo activity decrease was the 1st day – in the groups with isolated iri or isolated blood loos or isolated trauma (eg1, eg2, ef4) – and the 3rd day – in the groups with combined effects – isi with blood loss (eg3) and iri with mechanical trauma (eg5). present study suggests that, something caused peroxidation of lipid membranes of hepatocytes, e.g. toxins and hypoxia. in previously published results, the active response of the liver antioxidant system was represented by increased and subsequently depleted activity of antioxidant enzymes [34]. this studies coincide with other authors’ results. orlova e. et al. estimated that sod activity is highest in the liver and advised a correction of its insufficiency with “vin-vita” [35]. liver tissue damage as a result of application of tourniquet signalled the possibility for development of multiple organ failure due to the ischemiareperfusion limb syndrome [36, 37]. to sum up the above, the highest increase of malonic dialdehyde rate was observed against the background of a combination of haemostatic tourniquet on the thigh with blood loss. additionally, malonic dialdehyde content was higher in the group with a combination of trauma and the tourniquet in comparison with isolated mechanical trauma. this fact confirms the role of the tourniquet as a factor that complicates the course of traumatic disease due to the development of the ischemia-reperfusion syndrome. all of our interventions caused hypoxia – as a result of isolated bleeding and cessation of blood flow due to haemostatic seemedj 2020, vol 4, no. 2 liver peroxidation and limb ischemia-reperfusion injury 8 southeastern european medical journal, 2020; 4(2) tourniquet. among the causes that activated pathological mechanisms of ischemiareperfusion injury, attention should be paid to the influences of toxic derivatives of rhabdomyolysis, which entered the bloodstream in excessive concentrations after limb release from tourniquet. intensification of the liver function was the result of a protective response to pathogenic effects. the knowledge gained is very important, as in recent years the number of situations accompanied by bleeding from large blood vessels increased significantly. in addition, the use of haemostatic tourniquets is still one of the most efficient and easy way to control blood loss. thus, their importance cannot be denied, but side effects and their pathogenic effects on the systemic level require more detailed research. conclusion intensification and decrease of liver function was the result of a protective response to pathogenic effects. periods of lipid peroxidation activity allowed us to find 2 periods of exhaustion of wounded organisms against the background of blood loss, combined with the use of haemostatic tourniquets and consequently affected by ischemia-reperfusion syndrome – they are on the 3rd and 14th day against the background of bleeding combined with irs. knowledge of this will help to better understand the pathogenesis of traumatic disease and with a new form of oxidative stress and development of the best treatment. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. kauvar ds, dubick ma, walters tj, kragh jj. systematic review of prehospital tourniquet use in civilian limb trauma. j trauma acute care surg. 2018; 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[in russian]. 32. malinoski dj, slater ms, mullins rj. crush injury and rhabdomyolysis. crit care clin. 2004;20(1):171-192. doi:10.1016/s07490704(03)00091-5 33. behn c, arenada of, llanos aj, caledon g, gonzales g. hypoxia-related lipid peroxydation: evidences, implications and approaches / respiratory physiology and neurobiology. 2007. v. 158(2-3):143-150 doi 10.1016/j.resp.2007.06.001 34. volotovska nv, zarichna oy, kuzmak ip. aktyvnist katalazy ta superoksyddysmutazy na tli eksperymentalnoi ishemii-reperfuzii kintsivky [сatalase and superoxide dismutase activity on the background of experimental ischemiareperfusion of the limb] shpytalna khirurhiia. zhurnal imeni l. ya. kovalchuka – hospital surgery. l. ya. kovalchuk mag. 2019;2:53-59. doi 10.11603/2414-4533.2019.2.10418. 35. orlova ea, lazarchuk oa. aktivnost tsitozolnoy superoksiddismutazyi v tkanyah kryis raznogo vozrasta na fone primeneniya parafarmatsevtika «vin-vita». ukrayinskiy zhurnal klinichnoyi ta laboratornoyi meditsini. 2010; 5(3):87-90 [in russian]. 36. volotovska nv, kashchak tv antioxidant enzymes activity in experimental ischemiareperfusion injury. international journal of medicine and medical research. 2019;5(2): 8490. seemedj 2020, vol 4, no. 2 liver peroxidation and limb ischemia-reperfusion injury 11 southeastern european medical journal, 2020; 4(2) 37. tsymbaliuk hiu. stan dobovoho diurezu nyrok v umovakh ishemichno-reperfuziinoho syndromu kintsivok, travmy orhaniv cherevnoi porozhnyny, uskladnenoi hipovolemichnym shokom, ta yikh poiednannia u rannomu periodi travmatychnoi khvoroby. zdobutky klinichnoi i eksperymentalnoi medytsyny. 2018;3:163-169 [in ukrainian]. i author contribution: single author article i seemedj 2017, vol 1, no. 1 a cross-talk between the renin-angiotensin and adrenergic systems… 90 southeastern european medical journal, vol 1, 2017. a cross-talk between the renin-angiotensin and adrenergic systems in cardiovascular health and disease 1 ana stupin1, ines drenjancevic1, lidija rasic1, anita cosic1, marko stupin1,2 1 department of physiology and immunology, faculty of medicine josip juraj strossmayer university of osijek, osijek, croatia 2 department for cardiovascular diseases, osijek university hospital, osijek, croatia corresponding author: ana stupin, md, phd cavka.ana@gmail.com, anacavka@mefos.hr introduction this review aimed to present the interactions between the renin-angiotensin system (ras) received: april 19, 2017; revised version accepted: may 8, 2017; published: may 9. 2017 keywords: renin-angiotensin system, sympathetic nervous system, cardiovascular function, chronic heart failure, chronic kidney disease, salt intake and the sympathetic nervous system (sns) in health and diseases (e.g. cardiovascular, renal), with a special focus on these two systems interaction during hs intake in healthy abstract it is well accepted that a number of cardiovascular (cv) and renal diseases are characterized by the long-term activation of both the renin-angiotensin system (ras) and the sympathetic nervous system (sns), which also contribute to the pathophysiology of structural and functional cv abnormalities as well as to the final clinical outcome. moreover, there is a growing body of conclusive evidence that these systems do not operate independently, but interact at different levels throughout the cv system. the mediation of renin release from juxtaglomerular epithelioid (jge) cells in kidney by sns is well established and accepted. on the other hand, in recent years it became evident that ras, by its main effect or angiotensin ii (ang ii), induces sns activity in various organs and tissues. thus, there is a growing effort to clarify pathophysiological mechanisms of interaction and a more evident mutual potentiation of these two systems in different pathological states. since it became evident that a high salt (hs) intake, which is a major risk factor for hypertension development, has a deleterious impact on vascular and endothelial functions (even in the absence of blood pressure changes), it became necessary to investigate and clarify the effect of hs loading on major regulating systems—ras and sns—precisely in healthy individuals. the present review aimed to summarize the interactions between the ras and sns in health and diseases (e.g. cardiovascular, renal), with a special focus on these two systems’ interaction during hs intake in a healthy normotensive population. (stupin a, drenjancevic i, rasic l, cosic a, stupin m. a cross-talk between the renin-angiotensin and adrenergic systems in cardiovascular health and disease. seemedj 2017;1(1);90-107) seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 91 southeastern european medical journal, vol 1, 2017. individuals. the mediation of renin release from jge cells in kidney by β1-adrenergic receptors activation is well established and accepted (1). however, there is a growing interest to understand how ras affects sns activity, and to evaluate whether these two systems potentiate each other’s effects. it became evident that ras, by its main effector, angiotenin ii (ang ii), increases sns activity in various organs and tissues (e.g. the central nervous system, the adrenal medulla, the sympathetic ganglia and the sympathetic nerve endings), and that this interaction is mediated mostly by the ang ii receptors type1 (at1 receptors) located at the sympathetic nerve endings (2). the suggested physiological feedback loop in ras-sns interrelation is summarized in figure 1. most of these findings were brought by studies investigating the activity of these two systems in hypertension, chronic heart failure and/or chronic kidney disease (3, 4). even though hs intake unequivocally suppresses ras activity, the effect of hs intake on sns activity that is independent of blood pressure changes is still not completely understood. the role of the renin-angiotensin system in the cardiovascular physiological control system the ras is one of the most important hormonal systems which plays a key role in the arterial blood pressure, tissue perfusion, and extracellular volume homeostatic regulation, as well as in the regulation of neuronal and endocrine functions related to cv control (5, 6). ras consists of a cascade of functional proteins and exhibits its effects through the effector molecule ang ii. in this cascade, the first is a release of an aspartyl protease called renin, which is synthesized and released from renal jge cells located in the afferent and efferent arterioles of the renal glomerulus (1). renin release is stimulated by various stimuli including decreased renal perfusion pressure (1), increased renal sympathetic nerves activity and decreased nacl delivery to the macula densa of the juxtaglomerular apparatus (5, 6). renin cleaves angiotensinogen, which is synthesized by hepatocytes, to form the inactive decapeptide angiotensin i (ang i) (7, 8). ang i is converted to the active octapeptide ang ii by angiotensin-converting enzyme (ace) and nonace pathways (9, 10). non-ace pathways include ang ii production viae.g. chymase, which can be manifested in hypertensive patients treated with ace inhibitors who have increased ang ii levels despite their therapy, a phenomenon called ‘angiotensin escape’ (11). to carry out its biological functions, ang ii binds to two specific and ubiquitous g-protein coupled receptors, ang receptor type i (at1) and type ii (at2) (12, 13).at1 receptors mediate most of the established physiological effects of ang ii including actions on cv system (vasoconstriction, increased blood pressure, increased cardiac contractility, vascular and cardiac hypertrophy), kidneys (sodium reabsorption in renal tubule, inhibition of renin release), adrenal cortex (aldosterone synthesis) and sns (1, 5, 6). at2 receptors are generally assumed to counteract the vasoconstrictor and growth-stimulatory actions of at1 receptors (14). there are several biologically active angiotensin metabolites, including angiotensin iii, angiotensin iv and angiotensin-(1–7), which stimulate the at2 receptors (but with low affinity), and/or newly discovered putative receptors (14, 15). the physiological relevance of these metabolites in various tissues is still under investigation. aldosterone is another effector molecule of the ras, whose synthesis and secretion is stimulated by the ang ii mediated activation of at1 receptors in the adrenal zona glomerulosa cells. aldosterone promotes sodium reabsorption, water retention, and potassium and magnesium loss and modulates blood pressure (16). circulating and local (tissue) ras generally, ras can be divided to the circulating and local (tissue) ras (17). however, it is very hard to differentiate these two systems because of their extensive overlap (18). the circulating ras implies renin produced in kidneys, which cleaves liver-derived angiotensinogen to generate ang i that is converted by ace into ang ii. on the other hand, a key feature of local ras is the local synthesis of ras components (e.g. seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 92 southeastern european medical journal, vol 1, 2017. angiotensinogen, ang peptides) independently of the circulating ras. at1 and at2 receptors are invariably locally synthesized and are components of both circulating and local ras systems (19). in recent years, it has been demonstrated that the local ras includes many other potential components that may contribute to tissue-specific mechanisms of ang peptide formation, and subsequently may participate in the pathophysiology of various diseases or contribute to the mechanisms that protect from tissue injury. these include the (pro)renin receptor(20), ang generation from ang-(1-12) that is independent of renin (21), intracellular (or intracrine) ras that may contribute to cv disease (22, 23), and at2 receptors and the ace2/ang-(1-7)/mas receptor pathway that may mediate therapeutic benefit in cv disease (22, 23). furthermore, several novel angiotensin peptides have been described, including angiotensin iv, angiotensin a, alamandine, and angioprotectin (22, 23), which are suggested to have the potential to contribute to disease or to protective mechanisms. the challenge of future research regarding the ras is to investigate to what extent these two components of the ras system are independent of each other, and what is the clinical relevance of the local ras in cv disease development and progression. abnormal renin-angiotensin system activity in development of cardiovascular diseases the ras also has a central role in mediating and regulating vascular adaptive processes (24, 25) and is a major regulatory system involved in the regulation of a myriad of cv processes. abnormal activity of the ras leads to the development of different cv diseases and events (24), and results in vasoconstriction, muscular (vascular and cardiac) hypertrophy and fibrosis (25). changed ras activity is involved in secondary hypertension, including renin-secreting neoplasms, renovascular figure 1. physiological feedback loop in renin-angiotensin system – sympathetic nervous system (ras-sns) interrelation. seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 93 southeastern european medical journal, vol 1, 2017. hypertension, malignant hypertension, pheochromocytoma and primary hyperaldosteronism, and by primary hypertension, the plasma renin activity (pra) depending on the particular case, can be high, normal or low (26). furthermore, numerous signaling pathways, including cell proliferation, hypertrophy and apoptosis, in response to ang ii are mediated by reactive oxygen species, and oxidative stress is deeply associated with the progression of cv disease (27). the ras, through its physiological effectors, plays a key role in promoting and maintaining inflammation, and has proinflammatory and profibrotic effects at cellular and molecular levels. inflammation is an important mechanism in the development and progression of cv diseases such as hypertension and atherosclerosis. a dysfunctional endothelium is leaky and facilitates migration of inflammatory cells into the vascular wall and stimulates smooth muscle cells to proliferation (28). ang ii upregulates nfκb and related inflammatory genes and activates endothelial and endocardial nadph oxidase, which plays a central role in the generation of reactive oxygen species (ros) in cv disorders (29). interestingly, in almost all of the above-mentioned states, along with the ras hyperactivity there is increased sns activity as well (30), which sets the potential interaction of these two systems in the pathogenesis of cv and renal diseases in the focus of recent studies in this field. the role of the sympathetic nervous system in the cardiovascular physiological control system activation of the sns has long been recognized as a manifestation of various cv diseases including hypertension and the clinical syndrome of heart failure (30). abnormal increase in the circulating plasma catecholamines level and increased muscle sympathetic nerve activity (msna) were some of the first documented evidences of increased sns activity in cv disease patients (30). still, even though increased sns activity was generally (systemically) manifested in those patients, recent evidences implicate that sns hyperactivity is not uniformly distributed through the body, but rather has regional distribution differences, with hyperactivity in some areas and modest or even absent activity in others. thus, it seems that this generalized effect of sns on the cv system was overestimated, and that a special focus of future studies should be set on its effect on individual organs and organ systems (31). sympathetic nervous system in central nervous system the regulation of sns activity in the cns may have a crucial role in the pathogenesis of different cv diseases. the main sympathetic activity-regulating nuclei in the cns are the paraventricular nucleus in hypothalamus (pvn), rostral ventrolateral medulla (rvlm) and nucleus tractus solitarius (nts) (32, 33). nts receives signals from the cardiopulmonary afferents, including baroreceptors and chemoreceptors, and has an indirect effect on of the neuronal activity of the rvlm (32, 33). in addition, when activated, nts leads to the pnv activation as well, which is a major integrative nucleus that can influence sns activity and extracellular fluid volume by producing antidiuretic hormone (adh), which also has its repercussion on the activity of the cv system (34). pvn sends signals to rvlm which contains sympathetic premotor neurons for the cv system, and participates in sns regulation via communication with the intermediolateral column of spinal cord (iml) (33, 35). these central sns pathways are a focus of recent studies, since it has become more evident that the abnormal sympatho-excitation of the central sns deteriorates renal and cv function in various diseases (e.g. chronic kidney disease, hypertension, heart failure) and contributes to disease progression (30). peripheral sympathetic nervous system peripheral effects of the sns on the cv system, such as short-term—as much as long-term— control of blood pressure, are quite well known. the terminals of sympathetic nerves release norepinephrine, which binds to α-adrenergic receptors on vascular smooth muscle causing seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 94 southeastern european medical journal, vol 1, 2017. vasoconstriction (36, 37). many functional studies have confirmed that cutaneous blood flow is regulated by the sns (38, 39). sympathetic activity increases cardiac output by releasing epinephrine and norepinephrine (40) and also has effect on heart rate, which is mediated by the release of norepinephrine from postganglionic fibers that innervates the whole heart. this effect is mediated via β-adrenergic receptors on the heart muscle cells (40). thus, a β-adrenergic blockade is an effective treatment for elevated blood pressure. the sns regulates short-term transitions in blood pressure by the arterial barorefex (41), but also has a significant role in long-term blood pressure regulation. studies have shown that the pharmacological blockade of sympathetic ganglions decreases blood pressure in both hypertensive and normotensive subjects (42). chronic carotid baroreceptor stimulation evokes long-term reduction in the sns and blood pressure in hypertensive patients (43). another important part of the sns is a muscle sns. muscle sns positively correlated with total peripheral resistance, but negatively with cardiac output (44). further, constriction of peripheral resistance vessels in the response to norepinephrine was lower in subjects with higher sns activity, indicating that healthy people with higher muscle sns had a second protective factor to prevent elevation in blood pressure (45). these findings may suggest that, in regulating the cv system to keep its function optimal, sns activity affects not only well known and established pathways, but also many others not yet fully comprehensible. the kidneys are important targets of sympathetic tone modulation in general, since the sns is one of several factors that influences the efficiency of the renal regulation of blood pressure. anatomical and physiological evidence has shown that the sns innervates jga cells, renal tubules and vasculature (46, 47).thus, changes in renal sns activity frequency mediate increases in urinary sodium and water excretion by regulating the renal tubular water and sodium reabsorption throughout the nephron, changes in renal blood flow and the glomerular filtration rate by regulating the constriction of renal vasculature, and changes in the activity of the ras by regulating the renin release from jga cells (46, 47). increased renal sns activity decreases renal blood flow and the glomerular filtration rate. jga cells in the kidney have β-adrenergic receptors, which make the kidneys important targets of renal sns activity and a place where it is generally accepted that the “sns meets the ras” (figure 1). sns activity in kidney increases renin release by activating jga β-adrenergic receptor, along with an increase in tubular sodium reabsorption, and decreases in renal blood flow and the glomerular filtration rate (48). furthermore, an additional effect on renal circulation is present via response to exogenous epinephrine and norepinephrine, with afferent arterioles showing higher sensitivity to the vasoconstrictive effect of circulating catecholamines than efferent arterioles (48). this reveals the importance and relevance of sns activity on the kidney's compensatory mechanisms and management of volume expansion and high salt intake, which precedes pathological conditions such as hypertension. the sns also affects the immune system by contributing to leukocyte activation and extravasation, inflammation, oxidative stress and the production of chemokines and cytokines (49). thus, it is evident that understanding of the sns modulatory effects on cv function implies its general (systemic), central and peripheral (individual organ or tissue) effects, and its interactions as well. while these individual effects of sns are mainly well understood, there are many intertwined pathways between different sns components in both health and disease, which make the effect of the sns on the cv system more complex to understand and thus a focus of most recent studies on this issue. seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 95 southeastern european medical journal, vol 1, 2017. interaction between the reninangiotensin system and the sympathetic nervous system in the pathogenesis of cardiovascular and renal diseases there are several cv and renal diseases characterized by both ras and sns activation in which these systems, beside their effect on blood pressure regulation, also contribute to the pathophysiology of both structural and functional cv abnormalities and contribute significantly to clinical outcome (4). it became evident that these systems do not operate independently, but interact at different levels throughout the cv system. thus, there is a growing effort to clarify the pathophysiological mechanisms of interaction and more evident mutual potentiation of these two systems in different pathological states, including chronic kidney disease, essential hypertension, heart failure, obesity, metabolic syndrome, etc. the renin-angiotensin system and sympathetic nervous system interaction in chronic kidney disease chronic kidney disease (ckd) is often characterized by enhanced activity of the ras and sns (4), which is summarized in figure 2a. it is considered that kidney ischemia represents a central stimulation for renin secretion and ras activation that subsequently increases sns activity. intravenous infusion of ang ii stimulates msna in humans, and even a small locus of injury in one kidney leads to hypertension associated with increased central sympathetic activity. ang ii can interact with the sns on different sites, in the kidney, in the cns and on peripheral sites, enhancing norepinephrine release from sympathetic nerve activity (50). when taken into account that increased sns figure 2a. enhancement of renin-angiotensin system activity by sympathetic nervous system and vice versa in cardiovascular and renal diseases (chronic kidney disease) seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 96 southeastern european medical journal, vol 1, 2017. activity enhances ras activation by releasing renin from jge, it is evident that there may be a reciprocal potentiation between these two systems in the development and progression of ckd. thus, kidney injury, which is generally characterized by an increased ras activity, can lead to high sns activity, hypertension and finally end organ damage (50). moreover, there is vast experimental evidence showing that increased sns activity contributes at several levels to the development of cv organ damage (4, 51, 52). thus, with these pathophysiological mechanisms in mind, it seems logical to hypothesize that ras inhibition would reduce sns activity in ckd. moreover, recent studies reported that a sns blockade in addition to ras inhibitor treatment might be beneficial in a selected patients group (51-53). the renin-angiotensin system and the sympathetic nervous system interaction in chronic heart failure chronic heart failure (chf) is a multi-factorial disease that presents the end result of various insults to the myocardium (e.g. ischemic heart disease) (3). the most significant hallmark of chf is the continuous interaction between the underlying myocardial dysfunction (e.g. decrease of cardiac output) and activated compensatory mechanisms in order to maintain blood pressure and organ perfusion. activation of the ras along with the sns plays a crucial role in the pathophysiology of chf, which is summarized in figure 2b. activation of the sns is considered a major compensatory mechanism in the development of chf, which may be due to the changes in peripheral baroreceptors and chemoreceptors reflexes, chemical mediators figure 2b. enhancement of renin-angiotensin system activity by sympathetic nervous system and vice versa in cardiovascular and renal diseases (chronic heart failure) seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 97 southeastern european medical journal, vol 1, 2017. that control sympathetic outflow and central integrative sites (33) (figure 2b). in recent years ang ii, no and/or pro-inflammatory cytokines were described as crucial mediators controlling sympathetic outflow. on the other hand, an increase in renin release in chf state is caused by at least two pathways including renal sympatho-excitation as well as a decrease in renal perfusion pressure (3, 33). all of these compensatory mechanisms are initially beneficial; however, they became counterproductive if sustained for a prolonged time (figure 2b). areas in rvlm and in hypothalamus mediate and increase sympathetic outflow in response to a microinjection of ang ii, which was inhibited after a central infusion of at1 blocker losartan (54-56). interestingly, this increment in central ras activation could be associated with an increased oxidative stress level in chf state (57, 58). the role of the central ras in the supporting formation of a vicious circle in the development and progression of chf is not limited to the cns. francis et al. reported that a central blockade of ace decreased renal sns activity, improved blunted baroreflex sensitivity, and normalized sodium consumption, urine sodium and urine volume in rats with chf (59). furthermore, in a myocardial infarction (induced by acute coronary artery ligation) rat model with transgenic deletion of angiotensinogen (rats which express an antisense rna against angiotensinogen), deterioration of chf was not as progressive as in control chf rats (60). taken together, it became evident that a hyperactive central ras is a contributor to global physiological changes as well as the cv dysfunction seen in chf. on the trail of these findings there is a growing trend to consider the use of both pharmacological and nonpharmacological therapy targeting the central ras in the treatment of chf (3). clearly, the described cross talk between the central ras and increased sns activity is only one potential mechanism explaining the regulation of sns activity in chf (figure 2b). changes in the renin-angiotensin system and sympathetic nervous system during high-salt loading it is generally accepted that increased dietary salt intake is associated with an increase in arterial pressure, resulting in hypertension, which makes dietary salt a leading cause for cv, cerebrovascular, and renal morbidity and mortality (61). various mechanisms were suggested to contribute to the development and progression of salt sensitive hypertension, including increased activation of the ras and elevated sns activity, well established hallmarks of arterial hypertension (62). however, in recent years it became more and more evident that hs dietary intake affects vascular and particularly endothelial functions even in the absence of changes in blood pressure (bp) (6365).thus, it should be taken into account that the concept of salt-sensitivity is not limited only to the effect of dietary salt modulation on bp, but to its effect on vascular function and the cv system in general as well (63). there is an overall consensus that hs intake, high levels of ang ii and increased sympathetic activity are all injurious to the cv system and play a role in a multitude of cv diseases (66). however, much less is known about the role of elevated salt intake on cv functions that are independent of arterial bp, especially in healthy humans. in clarifying the pathogenic sequence in which an hs diet is on one side and impaired cv function (e.g. endothelial dysfunction, hypertension) on the other, it is necessary to investigate the effect of salt loading on major regulating systems (e.g. ras, sns) in healthy individuals. high salt feedback on the renin-angiotensin system it has been demonstrated unequivocally in both humans and experimental animals that salt intake is inversely related to ras activation: low salt intake stimulates ras activity, and hs intake suppresses it (67). dietary salt intake modulation affects ras activity via at least four different pathways including: 1) the macula densa mechanism, which regulates renin release in response to changes in the renal tubular salt seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 98 southeastern european medical journal, vol 1, 2017. concentration; 2) salt-dependent changes in arterial bp; 3) circulating salt-dependent hormones, particularly atrial natriuretic peptide (anp); and 4) the sns (67). still, signal pathways that adjust renin synthesis and ras activity to changes in salt intake are still not completely understood. recent data suggest that macula densa mechanism is involved in adjustment of renin release in response to acute changes in salt loading (68, 69). still, its effect on ras activation during long-term changes in salt loading is less clear: it does not appear to have a function in this situation, but rather to modulate the general responsiveness of renin release (67, 70). furthermore, numerous studies have reported that salt-dependent regulation of ras can occur even in the absence of changes in arterial bp, especially in healthy individuals (64, 65, 71). thus, bp is not a general controller of salt-dependent regulation of the ras under normal conditions, but it can modulate renin synthesis when salt modulation provokes significant changes in bp levels. it has been shown that an increase in extracellular volume, induced by oral salt intake or intravenous saline infusion, is associated with elevated plasma levels of atrial natriuretic peptide (anp), which consequently induce natriuresis and vasorelaxation, raise glomerular filtration and have capacity to directly suppress renin release from jge cells (72). however, whether this mechanism is relevant in physiological condition in healthy humans or animals is still unclear. studies have reported that modest acute salt loading in both healthy humans and animals did not elevate anp levels and did not suppress plasma renin activity, indicating that ras inhibition is not dependent on anp (73, 74). thus, anp seems inessential for ras suppression during acute salt loading, and its role in longterm dietary salt modulation should be addressed in further investigations. a number of studies investigated the correlation between salt intake and local renal sympathetic activity, indicating an inverse relation between the salt intake and renal nerve activity. since reninproducing jge cells have β1-adrenoreceptors whose activation results in renin release, one of the possible mechanisms mediating ras inhibition by hs loading could be inhibition of local renal sns activity (75, 76). still, evidence indicating that renal nerves have a role as a mediator of the salt-dependent regulation of renin release and synthesis are insufficient. this indisputable connection between salt intake and ras activity indicates that the central role in mediating salt homeostasis within the body and its effect on cv, cerebrovascular or renal function belongs particularly to the ras. in recent years, it became more evident that, besides its crucial role in body fluid volume, electrolyte balance and blood pressure regulation, the normal function of the ras is critical for maintaining arteriolar structure, vascular reactivity and cardiovascular health in general (5). hs-induced increased oxidative stress (5) and impairment of vascular function (that is independent of bp changes) are related to low levels of ang ii and a normally functioning ras has a protective effect in the maintenance of vascular function (77). in contrast to numerous animal studies reporting that ras inhibition provoked an increased oxidative stress level and endothelial dysfunction, the effect of ras inhibition on the vascular function in a healthy normotensive human population was set in a focus of a very few recent studies. most of these studies have found that hs intake (ras inhibition) impairs the flow mediated dilation of the brachial artery in the absence of bp changes (65), which was likely associated with reduced vascular no bioactivity (78-80). furthermore, same deleterious effect of acute salt loading was observed in skin microcirculation as well (64, 65, 79). cavka et al. reported that one week of a low-salt diet with oral losartan (a selective at1 receptor inhibitor) administration led to a significant increase in plasma levels of the cyclooxygenase dependent vasoconstrictor thromboxane (txa2) without causing any changes in bp and/or skin microvascular blood flow responsiveness in young healthy women, suggesting that an at1 receptor blockade may play an important role in the regulation of a cyclooxygenase-dependent pathway of metabolism of arachidonic acid (81). seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 99 southeastern european medical journal, vol 1, 2017. high salt feedback onsympathetic nervous activity sodium retention is link between cv and renovascular diseases together with elevated sns activity (3, 4). interestingly, it still remains to be investigated how salt intake affects sns activity in healthy individuals in the absence of bp changes (does it inhibit or potentiate sns activity), and whether this effect is uniform for both local and systemic sns responses (figure 1). there is a paucity of studies investigating the effect of hs loading on systemic sns activity in physiological conditions, both in animal and human models. so far, studies in animal models (rabbits) have reported that hs alone had no effect on baseline bp, water intake or sns activity, but in combination with low-dose ang ii infusion hs provoked sympatho-excitation (82). hs intake induces the central sensitization of sympathetic circuits to result in exaggerated cv reflexes and an increase in bp variability in normotensive salt-resistant animals (83). furthermore, a 6-day hs diet increased the mean systolic bp, decreased heart rate, and increased vagal activity in healthy, normotensive women (age 40–70) (84). these very few studies suggest that changes in autonomic nervous system balance should be taken into account during ras modulation by salt intake, even in young healthy normotensive individuals. regarding potential mechanisms that mediate interaction between salt intake and sns activity, it is well known that acute increase of plasma osmolality of sodium concentration in both plasma and cerebrospinal fluid can cause an increase of sns activity (85, 86), which is presumably mediated by osmosensory neurons in the organum vasculosum laminae terminalis (ovlt) in the brain (86). the most recent study by kinsman et al. has demonstrated that ovlt neurons are activated by modest rises in plasma or cerebrospinal nacl, and their activation elevates bp and triggers a pattern of sns activity that presumably facilitates renal sodium excretion in sprague dawley rats (87). furthermore, many other agents, such as endogenous ouabain are involved in blood pressure (bp) elevation. elevated sodium concentrations in body fluids can induce the secretion of the ouabain by both the hypothalamus and the adrenals, suggesting that ouabain could have an important role in linking together central and peripheral hypertension occurrences (88). ouabain acts centrally in the brain to increase the sympathetic drive (89) and in the periphery causing vasoconstriction via myocytes and endothelium (90). still, when taken into account that in most studies (both animal and human) in which moderate hs loading provoked vascular dysfunction independently of bp changes, there was no significant increase in sodium concentration in plasma. the above suggested effects of the described conditions are disputable and remain in need of clarification. another intriguing relation is that between the sns and ras during hs intake. an inverse relation of hs intake and renal sns activity was observed (91, 92), showing that hs intake can suppress renal sns activity. renal sns activity also affects ras, whereas renal sns activity stimulates renin release via the activation of β1 adrenoceptors placed in renin-producing jge cells (75, 93), as already described. to examine this axis, studies using β1/β2 double knockout mice fed with a hs diet were performed (94). in this study β1/β2 double knockout mice were fed with a hs diet, and surprisingly they maintained the salt-dependent regulation of renin plasma concentration, since low salt intake stimulated renin release, and hs intake suppressed it (94). similarly, dogs were fed with a hs diet and the changes in sns activity effects were investigated via modulating baroreceptor activity (95). the suppression of central sns activity by electrical stimulation of baroreceptors lowered bp without affecting the ras and without impairing the normal saltdependent secretion of renin. this may suggest that neither renal nor central sns activity is crucial in the salt-dependent regulation of the ras, but surely acts as a moderator in the ras response to changes in salt load (67). since most organs possess a local ras that is regulated independently and is somewhat compartmentalized from the circulation, it has been suggested that the brain ras may play an important role in cv regulation through its ability seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 100 southeastern european medical journal, vol 1, 2017. to modulate sns activity (96) (figure 1). studies in animal models have shown that increased levels of brain ang ii and the activation of brain at1 receptors in the rvlm mediate the sympatho-excitatory actions (96). to date, the limited data indicate that elevated dietary salt intake enhances both sympatho-excitatory and sympatho-inhibitory responses evoked by a number of neurotransmitters exogenously applied to the rvlm (97-99), but these enhanced responses could not be attributed to changes in downstream sympathetic pathways or vascular reactivity (98-100). on the other hand, results obtained in studies on experimental animals have demonstrated that the brain ang ii activates posterior hypothalamic nuclei that increase efferent renal nerve activity and bp, suggesting the presence of renal/cerebral interaction in which the brain ras can regulate the peripheral sympathetic activity and renal ras (101). taken together, the relation between sns activity and salt intake and its role in the development and progression of hypertension is yet unclear. conclusions many separated parts and mechanisms of the ras and sns affecting the cv system are discovered and explained, but the interplay of these separated mechanisms seems to have a pivotal role in explaining and understanding this highly interactive network. studies conducted so far may suggest that some parts of the ras and/or sns effect on the cv system are overestimated, while others are underestimated. in a situation such as hs intake, both ras and sns activity may have a modulatory, compensatory role, and this role can increase as the cv and renovascular diseases progress. metabolic syndrome and a series of cardiovascular problems (35), it is very important that the values were within healthy limits. some researches show that, similar to bmi, values for pbf and whr rise as people age (36). the body composition (presented in table 1) of the subjects shows that they have appropriate mass of fat and muscles, and their body composition is in accordance to weight status based on bmi value. in other words, the prevalence of obesity based on bmi distribution would probably be similar if they were distributed according to the bf% or slm. results reported by grygiel-gorniak et al. (37) show similar values of bmi, whr and bf% with the results of the present study, as well as the similar differences between sexes. our findings regarding differences in bmi and bf% between men and women showed that men had higher bmi, but lower bf% compared to women. this could be ascribed to the greater muscle mass in men. however, the correlation between bf% and bmi showed a statistically significant high positive value, indicating a strong connection between those two variables. correlation was slightly lower in men than in women. there was also high correlation between bf% and whr in both sexes. collins et al., in their study of association of bmi and bf% among bmi-defined non-obese middle-aged individuals, found that the bmi category was not concordant with the %bf classification for 30% of the population. the greatest discordance between %bf and bmi was observed among %bf-defined overweight/obese women (38). a strong correlation of bmi and bf% in young women was reported in the study of bakir et al. (39). they obtained correlation coefficients between bmi and bf% of 0.74 for women aged 18-30 years. proportions of weight categories were not significantly different over the years in which measurements were made. students that choose to attend the college of applied sciences in vukovar are similar in weight status throughout the years. this result is different compared to predictions of increase in obesity prevalence, and shows a steady state in the weight status of first-year students during the years examined, without any increase of obesity. it is possible, however, that it might have been too short a period for potential trends to reveal themselves. based on the presented results, the conclusion could be made that most of the freshman students at the college of applied sciences fall in the category of normal weight, with an seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 101 southeastern european medical journal, vol 1, 2017. overweight prevalence of around 19-20%, including around 5% obese persons among them. there are also 6.5% of those who are underweight. there is a higher tendency toward the prevalence of overweight persons among men, while among women there is a higher tendency for underweight prevalence. the prevalence of obesity and the overall distribution across the weight categories, as well as the body composition of the first-year students have not changed during the period from 2008 to 2016. funding this review article was supported by the croatian science foundation under the project #ip-2016-06-8744 interaction of reninangiotensin and adrenergic system in oxidative stress induced endothelial activation (rasadrenox) and the croatian science foundation grant #ip-09-2014-6380 impaired vasorelaxation and endothelial leukocyte interaction (eli) in development of atherosclerotic lesions (v-eli athero). references 1. tobian l, tomboulian a, janecek j. the effect of high perfusion pressure on the 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2021, vol 5, no. 1 oral hygiene and disabilities 170 southeastern european medical journal, 2021; 5(1) review article importance of oral hygiene and maintaining oral health in persons with disabilities 1 martina juzbašić ¹,²*, davor seifert ¹,matej tomas ¹,², marija čandrlić¹,³, marko matijević¹ 1 faculty of dental medicine and health osijek, josip juraj strossmayer university of osijek, osijek, croatia ² interdisciplinary university study of molecular biosciences, josip juraj strossmayer university of osijek, osijek, croatia ³ faculty of medicine osijek, josip juraj strossmayer university of osijek, osijek, croatia # co-editor of the manuscript: s. lecturer mirna sabljar, phd; the academy of arts and culture in osijek, university of osijek, croatia corresponding author: martina juzbašić, martina.juzbasic@fdmz.hr received: sep 7, 2020; revised version accepted: feb 2, 2021; published: apr 28, 2021 keywords: oral health, oral hygiene, persons with disabilities, dental plaque abstract this paper aims to approximate and facilitate the routine of daily oral hygiene for persons with disabilities, as well as to emphasize the importance of educating persons with disabilities and their caregivers about oral health as an essential part of overall health. desk research of electronic databases was conducted with the aim of writing this paper, using the following keywords: ‘oral hygiene’, ‘dental plaque’, ‘oral health’ and ‘persons with disabilities’. literature research has shown that persons with disabilities have poor oral hygiene, as well as that there is a lack of education among them and their caregivers about the importance of oral health and proper oral hygiene. poor oral hygiene can affect a person’s quality of life due to discomfort during eating, bad breath, poor selfesteem, pain, and disturbed sleep, which is a result of caries or other diseases of the oral cavity. maintaining oral health is an essential part of overall health. (juzbašić,m, seifert d, tomas m, čandrlić m, matijević m. importance of oral hygiene and maintaining oral health in persons with disabilities. seemedj 2021; 5(1); 170-175) seemedj 2021, vol 5, no. 1 oral hygiene and disabilities 171 southeastern european medical journal, 2021; 5(1) introduction it is estimated that around one billion people, which is about 15% of the world’s population, have some form of disability. the term disability is broad and covers persons with physical, sensory, intellectual, medical, emotional and social disorders, and most commonly a combination of these disorders (1). persons with disabilities often need extra help to achieve and maintain good health, and oral health is no exception. oral hygiene is the process of cleaning the hard and soft tissue of the oral cavity (teeth, gums, and tongue), fixed and removable prosthodontic restorations, and dental appliances. irregular or poor oral hygiene results in an increased number and variety of bacteria in plaque (2). dental plaque plays a significant role in the development of the two most common oral diseases, dental caries and periodontal disease. most toothaches are caused by caries. at first, caries looks like a painless white stain; at this stage, the process is reversible with good hygiene, and remineralization of the enamel is possible. with the advancement of demineralization, cavitation and caries coloration occur. if left untreated, caries progresses to the pulp and causes inflammation accompanied by severe pain. inflammation can then spread to the top of the tooth, forming a granuloma, abscess, or cyst (2). persons with disabilities mostly have genetic predispositions for the development of gingival and periodontal disease, and the medications that they take, in combination with poor oral hygiene, inevitably lead to rapid disease progression, gum recession, and tooth loss (3, 4). it is important to raise awareness among persons with disabilities and their caregivers about the importance of maintaining oral hygiene and ways to achieve it, with emphasis on the need for regular visits to the dentist, who will teach them about routine and special procedures. dental plaque dentobacterial plaque is a soft deposit of living and non-living microorganisms in a matrix rich in polysaccharides and glycoproteins, which adheres tightly to the tooth surface and can only be removed by mechanical cleaning. the formation of dentobacterial plaque is a complex process that occurs in several stages that are not restricted. initially, a pellicle is formed, the first acquired plaque on the teeth. the pellicle is a thin, translucent glycoprotein layer with no cells and bacteria. it is 10 µm thick and forms 20 minutes after the tooth has been cleaned. once the pellicle has formed, bacteria begin to settle. bacteria can adhere to the pellicle: through direct contact between the bacterial wall and the pellicle, through fibrous extensions on the walls of the bacteria, through mediation of a fluffy layer of epithelial cells. at this stage, the plaque matrix is still sufficiently permeable, and aerobic conditions prevail (5). gram-positive streptococci such as s. mutans, s. sanguinus, s. oralis, s. mitis, and neissera spp. are the first to colonize the tooth surface. the primary colonizers of dentobacterial plaque are either aerobes or facultative anaerobes. secondary colonizers are mostly gram-negative bacteria such as actinomyces spp., fusobacterium, prevotella intermedia, and capnocytophaga spp. (6). from the third to the seventh day, the formation of extracellular polysaccharides dextran, mutan and levan intensifies. they increase the volume of plaque and reduce its permeability. only small molecules such as sucrose, to which great cariogenic potential is attributed, can now penetrate the plaque. due to the lack of oxygen, anaerobic conditions occur, in which the final products of decomposition of sucrose are pyruvic acid and lactic acid. the ph values are lowered to critical values of 5.2 to 5.4 (5). if dental plaque remains undisturbed for about seven days, tertiary colonizers accumulate. these are mostly strict anaerobes that opportunistically exploit the environment provided by other bacteria, including pathogenic bacteria such as porphyromonas gingivalis, aggregatibacter actinomycetemcomitans, and seemedj 2021, vol 5, no. 1 oral hygiene and disabilities 172 southeastern european medical journal, 2021; 5(1) spirochetes such as treponema denticola. when living as a microbial community in dentobacterial plaque, individual bacteria tend to share their virulence properties through gene transfer, particularly antibiotic resistance genes found in plasmids (6). the pathogenic biofilm of dental plaque results not only in dental diseases such as dental caries and periodontal disease, but may also be involved in the development of cardiovascular, respiratory and renal diseases, as well as diseases of other organs (6). it is therefore important to understand the mechanism of occurrence and the possibilities of prevention and control of dentobacterial plaque. oral hygiene in persons with disabilities persons with disabilities usually have poorer oral health, more extracted teeth, more caries, fewer fillings and treated teeth, gingivitis, a higher rate of edentulism, fewer preventative procedures, and more inadequate dental care compared to the general population (7, 8). as oral diseases primarily have a microbiological origin, it is necessary to establish plaque control, which can be achieved mechanically and chemically (9). the essential means for removing plaque from teeth are mechanical brushing agents and interdental cleaners such as dental floss and interdental brushes (10). use of soft toothbrushes as well as modified and electric toothbrushes is recommended, especially for persons with reduced motor skills and intellectual abilities. studies have shown greater plaque removal efficacy in this population when electric toothbrushes are used (11). it is equally important to clean the spaces between the teeth, so it is essential to educate patients, in line with their perceptual and cognitive abilities, about the use of interdental brushes and dental floss. most of these patients will find it more convenient to use dental floss with a holder (12). in addition to mechanical cleaning, chemical control of plaque is of great importance. it includes the use of toothpaste, gels, mouthwashes, etc., the composition of which contains active ingredients such as fluorides and chlorhexidine. various studies have demonstrated the efficacy of using chlorhexidine in persons who are unable to maintain proper oral hygiene mechanically, and it is the first choice for supporting the treatment of gingivitis and periodontal diseases (13). adequate use of fluoride is recommended from an early age, because of the high risk of caries in this population. where lower risk is assessed, the recommendation is to use a toothpaste with up to 1000 ppm fluoride for young children, and with around 1500 ppm after the age of 6. in case of high risk, which pertains to most children with disabilities, toothpaste or gel with 1000 to 1500 ppm fluoride is administered immediately when the first teeth appear in the mouth (pea-size). for 12-year-old children at high risk, 2800 ppm of fluoride is recommended, and up to 5000 ppm at the age of 16 (14). education of persons with disabilities and their caregivers persons with disabilities and their caregivers need to be educated about and conscious of the importance of oral health: how to achieve and maintain it. despite evidence that this population generally has poor oral health, oral hygiene training programs are not readily available for their caregivers (15). the results of the research show an improvement in the knowledge, skills, and attitudes of caregivers about oral hygiene after completing an educational program (15, 16). education about oral hygiene and dental cleaning procedures should be provided from an early age in order for children to develop the habit of maintaining oral hygiene and oral health. to support this, one study conducted among children with disabilities of preschool age showed a significant correlation of good tooth brushing with adaptive skills and practicing oral hygiene. multiple oral hygiene steps could be performed by children who developed the habit of brushing their teeth before the age of 1 and who regularly brushed their teeth at least twice a day, as opposed to children who started with oral hygiene later and brushed their teeth only occasionally (17). since one type of education does not suit everyone, such education needs to seemedj 2021, vol 5, no. 1 oral hygiene and disabilities 173 southeastern european medical journal, 2021; 5(1) be tailored to the specific condition of the individual or group. recent research has proved the importance of tailor-made education for achieving significantly better results in regard to understanding oral health and oral hygiene. a study conducted among the deaf and hard of hearing showed that participants with standard education in print form achieved good results compared to their pre-education status. however, a group of participants with tailor-made training, which included a video in sign language, showed statistically significant results when compared to their initial knowledge and skills in oral hygiene (18). it is crucial to regularly visit the chosen doctor of dental medicine, who will monitor oral health and provide additional oral hygiene instructions depending on the psycho-physical capabilities of the individual patient. for example, a dentist will demonstrate the process of tooth brushing to blind and partially sighted people using the ‘hand-to-hand’ technique, guiding the patient’s hand, alerting them to particular locations in the mouth that have a more significant physiological tendency to accumulate plaque, such as areas of contact between tooth and gums, gritted and rotated teeth, etc. (12). other methods, also used with the general population in the event of higher anxiety when performing dental procedures, help reduce fear in persons with disabilities. one of them is the so-called ‘tellshow-do’ method, in which, before any procedure is performed, it is explained to the patient using understandable language, and instruments that will be used are shown. it always begins with a tool or procedure that will cause the least fear in patients (13, 19). the doctor of dental medicine will apply various other methods in communication, preventive and curative action, depending on the patient’s medical history and individual needs. regular visits to the dentist and intervention training decrease dental phobia and aversion to the doctor’s office, as well as to necessary preventive and curative treatments (20). the importance of healthy and balanced nutrition, which is an essential factor in oral health, should undoubtedly be included in the education of persons with disabilities and their caregivers. for example, this could include highlighting the negative role of sugar in the onset of dental disease and pointing out some ingredients in the diet that have a potentially anticarcinogenic effect, such as xylitol (13, 21). sedation and general anaesthesia most persons with disabilities can be treated routinely in dental practices, with procedures tailored to them. however, some of them cannot cooperate well enough for the necessary dental procedure to be performed (22). appropriate sedation or general anaesthesia is used on all patients who are unable to cooperate during a visit to the dental office: very young children, patients with motor or cognitive dysfunctions, general population with severe dental phobia; this is likewise used in cases when procedures are extensive and cannot be done under local anaesthesia (23, 24). today, sedation is most often performed using an oral sedative or inhalation of nitric oxide. these methods can be very effective in relaxing anxious patients, but require a certain dose of cooperation; e.g., for inhalation, the patient must be willing to wear a mask through which gas is inhaled and must breathe through the nose (22, 25). general anaesthesia involves a reversible loss of consciousness caused by the use of one or more anaesthetics, during which the patient cannot be awakened by painful stimulation. it does not require patient cooperation and its application is considered relatively safe and has been widely described as a useful way of treating patients with a disability (22, 26). general anaesthesia allows for complete oral rehabilitation in the same visit, including tooth extraction, pulp and root canal treatment, placement of necessary tooth fillings, treatment with fixed and removable prosthodontic restorations, scaling and root planing, prophylactic procedures, etc. (27). seemedj 2021, vol 5, no. 1 oral hygiene and disabilities 174 southeastern european medical journal, 2021; 5(1) conclusion poor oral hygiene and diseases of the teeth and oral cavity limit normal daily functions such as eating, swallowing, chewing, and ultimately result in poorer general health. the impact of oral diseases on systemic diseases and the functioning of the organism in this specific population plays a significant role, even more so than in the general population (13, 28). therefore, a doctor of dental medicine should be an indispensable part of a multidisciplinary team that looks after the health of persons with disabilities. society should also contribute to better quality of life and the realization of optimal healthcare by providing high-quality health insurance, more frequent visits to the dentist, and more preventative and curative procedures, which are key to maintaining oral health (14, 29). oral disease prevention as an integrative part of overall health should be imperative, with emphasis on the mandatory continuous education of persons with disabilities or their caregivers, good oral hygiene, which includes mechanical and chemical plaque control, regular visits to the dentist, and proper nutrition. good oral health improves the quality of life. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. fdi world dental federation. oral health and dental care of people with disabilites. available at: https://www.fdiworlddental.org/resources/po licy-statements-and-resolutions/oral-healthand-dental-care-of-people-with-disabilities. date of access: 25.11.2019. 2. holt r, roberts g, scully c. dental damage, sequelae, and prevention. west j med. 2011; 174(4):288-90. 3. ferreira r, michel rc, greghi sl, et al. prevention and periodontal treatment in down syndrome patients: a systematic review. plos one. 2016; 11(6). 4. caton jg, armitage g, berglundh t, et al. a new classification scheme for periodontal and peri-implant diseases and conditions introduction and key changes from the 1999 classification. j periodontol. 2018; 89(suppl 1):18. 5. valm am. the structure of dental plaque microbial communities in the transition from health to dental caries and periodontal disease. j mol biol. 2019; 431(16):2957-2969. 6. seneviratne cj, zhang cf, samaranayake lp. dental plaque biofilm in oral health and disease. chin j dent res. 2011; 14(2):87-94. 7. wilson nj, lin z, villarosa a, george a. oral health status and reported oral health problems in people with intellectual disability: a literature review. jidr. 2019; 44(3):292-304. 8. peerbhay f, titinchi f. dental management of children with special healthcare needs. sadj. 2014; 69(5):214-20. 9. mcgrath c, zhou n, ming wong h. a systematic review and meta‐analysis of dental plaque control among children and adolescents with intellectual disabilities. jarid. 2019; 32(3):522-32. 10. ansari g, torabzadeh h, nabavi zs, hassani ps. comparing the effect of dry and wet brushing on dental plaque removal in children. j indian soc pedod prev dent. 2019; 37(3):292-6. 11. cem doğan m, alaçam a, aşici n, odabaş m, seydaoğlu g. clinical evaluation of the plaque‐removing ability of three different toothbrushes in a mentally disabled group. acta odontol scand. 2004; 62(6):350-4. 12. nenadić k, šubarić ž, dumančić j. persons with visual impairment-our patients. guidebook for interaction with blind and visually impaired persons for medical personnel. hrvatski savez slijepih, stomatološki fakultet sveučilišta u zagrebu, zagreb 2015. available at: https://www.sfzg.unizg.hr/_download/reposit ory/osobe_sa_ostecenjima_vida__nasi_pacijenti_vodic_za_zdravstvene_djelatnik e_6mb_2018.pdf. date of access: 25.11.2019. seemedj 2021, vol 5, no. 1 oral hygiene and disabilities 175 southeastern european medical journal, 2021; 5(1) 13. ajay b, manish j, sudhanshu s, sonal k. oral health preventive protocol for mentally disabled subjects– a review. j. adv dental research 2011; 3(1): 21-6. 14. dumančić, j. djeca s posebnim potrebama u stomatološkoj ordinaciji. dječja dentalna medicina. zagreb. naklada slap 2015: 407-14. 15. fickert na, ross d. effectiveness of a caregiver education program on providing oral care to individuals with intellectual and developmental disabilities. intellect dev disabil. 2012; 50(3):219-32. 16. mac giolla phadraig c, guerin s, nunn j. train the trainer? a randomized controlled trial of a multi-tiered oral health education programme in community-based residential services for adults with intellectual disability. community dent oral epidemiol. 2013; 41(2):182– 92. 17. zhou n, wong hm, mcgrath c. the impact of adaptive functioning and oral hygiene practices on observed tooth-brushing performance among preschool children with special health care needs. matern child health j 2019; 23:1587–94. 18. fageeh hn, mansoor ma. the effectiveness of oral hygiene instructions in sign language among hearing impaired adults in saudi arabia. spec care dentist. 2020; 40(1);41-8. 19. american academy of pediatric dentistry clinical affairs committeebehavior management subcommittee. guideline on behavior guidance for the pediatric dental patient. pediatr dent. 2008-2009 ;30(7):125-33. 20. yilmaz s, ozlü y, ekuklu g. the effect of dental training on the reactions of mentally handicapped children's behavior in the dental office. asdc journal of dentistry for children. 1999; 66(3):188-191, 154-5. 21. hayes c. the effect of non-cariogenic sweeteners on the prevention of dental caries: a review of the evidence. j dent educ. 2001; 65(10):1106-9. 22. dougherty n. the dental patient with special needs: a review of indications for treatment under general anesthesia. spec care dentist 2009; 29(1): 17-20. 23. wang yc, lin ih, huang ch, fan sz. dental anesthesia for patients with special needs. acta anaesthesiol taiwan. 2012; 50(3):1225. 24. brailo v, janković b, lozić m, et al. dental treatment under general anesthesia in a day care surgery setting. acta stomatol croat. 2019; 53(1):64-71. 25. dougherty n, romer m, perlman sp. the use of sedation for the dental patient with special needs. exceptional parent magazine. 2002; 113–5. 26. lim mawt, borromeo gl. the use of general anesthesia to facilitate dental treatment in adult patients with special needs. j dent anesth pain med. 2017; 17(2):91-103. 27. lee py, chou my, chen yl, chen lp, wang cj, huang wh. comprehensive dental treatment under general anesthesia in healthy and disabled children. chang gung med j. 2009 ;32(6):636-42. 28. stiefel dj. dental care considerations for disabled adults. spec care dent. 2002; 22(3):2639. 29. szylagyi pg, health insurance and children with disabilities. future child. 2012; 22(1):123-48. author contribution. acquisition of data: juzbašić,m, seifert d, tomas m, čandrlić m, matijević m administrative, technical or logistic support: juzbašić,m, seifert d, tomas m, čandrlić m, matijević m analysis and interpretation of data: juzbašić,m, seifert d, tomas m, čandrlić m, matijević m conception and design: juzbašić,m, seifert d, tomas m, čandrlić m, matijević m critical revision of the article for important intellectual content: juzbašić,m, seifert d, tomas m, čandrlić m, matijević m drafting of the article: juzbašić,m, seifert d, tomas m, čandrlić m, matijević m final approval of the article: juzbašić,m, seifert d, tomas m, čandrlić m, matijević m seemedj 2020, vol 4, no. 2 thyroid disease in pediatric population 77 southeastern european medical journal, 2020; 4(2) original article characteristics of thyroid disease in pediatric population 1 anja tomic *1, silvija puseljic 1,2, visnja tomac 1,2 1 faculty of medicine, university of osijek, croatia 2 university hospital centre osijek, pediatric clinic *corresponding author: anja tomic, anja.tomic93@gmail.com received: mar 8, 2020; revised version accepted: oct 5, 2020; published: nov 12, 2020 keywords: thyroid disease, pediatrics, incidence, autoimmunity abstract aim: in the last decade, an increase in the incidence of autoimmune thyroid disease in children has been observed. the aim of the study was to determine the incidence of thyroid disease in the pediatric population treated at the pediatric clinic in osijek; to determine if there was a rise in thyroid disease incidence in pediatric population in the observed period; and to determine whether there was any association of certain factors with an increased incidence of thyroid disease. methods: this study was a two-year retrospective study in two separately observed one-year periods (2010 and 2017). all patients with thyroid endocrinological disorders treated at the pediatric clinic of the university hospital centre osijek were included in the study. their medical records were used to obtain data. differences between variables were tested by the χ2 test, fisher's exact test and mann-whitney u test. the level of statistical significance was accepted for p <0.05. results: there was a 2.4-fold increase in the approximate incidence of thyroid disease, 9.7-fold higher approximate incidence of autoimmune thyroiditis, 9.75-fold higher approximate incidence of unspecified hypothyroidism and a triple increase in the approximate incidence of non-toxic goiter. other unspecified thyroid diseases are also statistically significantly on the rise. an analysis of factors such as anamnestic features, age, gender, comorbidities and other relevant data could not explain the increased incidence of thyroid disease. conclusion: the incidence of thyroid disease has increased considerably in the observed years, following world trends. efforts should be taken to clarify the mechanisms and factors involved in causing thyroid diseases in the pediatric population. (tomic a, puseljic s, tomac v. characteristics of thyroid disease in pediatric population. seemedj 2020; 4(2); 77-87) seemedj 2020, vol 4, no. 2 thyroid disease in pediatric population 78 southeastern european medical journal, 2020; 4(2) introduction the normal thyroid secretes thyroid hormones triiodothyronine (t3) and thyroxine (t4), which are crucial for normal growth and development, body temperature and regulation of energy levels. the basic functions of the thyroid hormones are regulation of oxidation in cells, stimulation of oxidative processes in which heat is generated, stimulation of protein synthesis, stimulation of growth and differentiation and acceleration of the metabolism of carbohydrates, fats and vitamins (1). proper thyroid function is essential for early neurocognitive development, growth and development during childhood and adolescence (2). optimal concentration of thyroid hormones is crucial, since a prolonged hypothyroid condition can result in a variety of disorders. hypofunction of the thyroid gland occurring before the age of 2 usually leads to delayed maturation of the skeleton and the development of intellectual disability (1). on the other hand, hypothyroidism occurring after a period crucial for neurocognitive development can lead to a slowdown in linear growth and maturation of the skeleton, with no effect on cognitive development and obesity development (3, 4). furthermore, hypothyroidism diagnosed in the late prepubertal years, which usually happens due to hashimoto's thyroiditis, can delay the onset of puberty or lead to premature isosexual pseudopuberty (development of breasts and internal reproductive organs in girls and increased testicular volume without adrenarche in boys). later in adolescence and young adulthood, hypothyroidism can lead to menstrual disorders, infertility and miscarriages (5, 6). several dozens of diseases have an autoimmune origin and such diseases affect 5-7% of the world's population (7). thyroid disorders are one of the most common endocrinological problems in children and adolescents. autoimmune thyroid disease is estimated to be the most common organospecific autoimmune disease, affecting up to 5% of the general population and 0.3 to 9.6% of the pediatric and adolescent population (8-10). epidemiological studies have suggested an increase in the incidence of autoimmune diseases in western societies over the last few decades (11). the rise in the incidence of autoimmune diseases raises the question of the factors that perpetuate it. given a decreasing frequency of infections in western countries, with an increase in the incidence of allergic disorders and neoplasms, which is accompanied by an increase in the incidence of autoimmune diseases, a hygiene hypothesis has been put forward (12). concordance of autoimmune disease in identical twins is 12-67%, which also highlights the potential role of epigenetics in disease development (13). over the last few decades, there have been significant changes in nutrition, the environment and exposure to pollution, environmental infections and stress management. another study highlights the significance of tobacco smoking, psychological distress, iodine intake, intrauterine development, bacterial and viral infections and medications such as interferon (14). therefore, with the previously established contribution of genetics, increasing attention is shifted on environmental factors and the lifestyle in western countries (15). the link between the causative factors of autoimmune disease – genetics and the environment – has not yet been fully elucidated. although much progress has been made in understanding the pathogenesis of autoimmune diseases, many questions remain unanswered. numerous studies conducted both in clinical settings and in experimental models have shown that changes in posttranslational histone modifications and dna methylation – two major epigenetic mechanisms – could potentially cause immune tolerance breakdown and the development of autoimmune diseases (16, 17). therefore, autoimmune diseases actually reflect the complex interactions between gene variation and the environment (18). in most cases, the origin of the stimuli leading to epigenetic changes in patients with autoimmune diseases remains undefined, but may include different external (e.g. diet, sun exposure, chemicals, environment, medicines) and internal (e.g. aging, stress, sex hormones) stimuli. xlinked genes are typically unmethylated (active) seemedj 2020, vol 4, no. 2 thyroid disease in pediatric population 79 southeastern european medical journal, 2020; 4(2) in males, while females have one methylated and one unmethylated gene. several molecules encoded on the x chromosome have also been found to play a significant role in the development of autoimmune diseases (7). an epigenetic link between diet and autoimmune diseases has also been observed, since certain foods provide donors of methyl groups (methionine, choline) and cofactors (folic acid, vitamin b12 and pyrodoxal phosphate), which are essential for dna and histone methylation (19). in addition to nutrition, ultraviolet radiation has been shown to stimulate epigenetic changes involving the hypermethylation of numerous promoter genes, some of which have an immunosuppressive effect (20). furthermore, studies conducted on twins have shown that, although identical twins are epigenetically minimally different at a young age, they happen to show numerous differences in dna methylation and histone acetylation later in life (21, 22). taken together, epigenetics has become increasingly important in the study of the etiopathogenesis of the entire spectrum of diseases, especially autoimmune disorders, and any new insights will be of great importance for a better understanding of this growing health problem. the objectives of present study were to determine the incidence of thyroid disease in the pediatric population treated at the pediatric clinic in osijek; to determine if there was a rise of thyroid disease incidence in pediatric population in 2010 and 2017; and to determine whether there was any association of certain factors, such as anamnestic features, gender, comorbidities and other relevant factors, with an increased incidence of thyroid disease in childhood for a given period. patients and methods all pediatric patients treated at the pediatric clinic of the university hospital centre osijek within the periods from january 1st 2010 to december 31st 2010 and from january 1st 2017 to december 31st 2017 who were monitored for thyroid disorders were included in the study. their medical records were used to obtain data. the data collected from medical records included age, sex, diagnoses of thyroid disease, status of thyroid hormones, presence/absence of thyroid gland specific anti-bodies, comorbidities, components of physical status and family history data. during a follow-up period, the same pediatric specialist and pediatric endocrinology subspecialist monitored and guided the patients. the criteria for inclusion of the subjects in the research were as follows: patient under the age of 18 treated at the pediatric endocrinology outpatient clinic in the periods from january 1st 2010 to december 31st 2010 and from january 1st 2017 to december 31st 2017; patient has one of the following diagnoses of thyroid disease (revision 10 of the international classification of diseases and related health problems): congenital hypothyroidism e03.0 / e03.1, other unspecified hypothyroidism e03.9, non-toxic goiter e04, hyperthyroidism / thyrotoxicosis e05, autoimmune thyroiditis e06.3, unspecified thyroid disorder e07.9, postprocedural hypothyroidism e89.0, malignant thyroid neoplasm c73, and observation z03. based on the data of the total population in the republic of croatia, the incidence of new pediatric thyroid disease cases was estimated. since there were no subjects over 14 years of age (specifically, in both years, the oldest ones were 12 years old), the data from the population register for ages 014 were used (24). due to the dynamics of population change and the inability to present current population values, the approximation of the population incidence was expressed at 95% confidence intervals. the study was approved by the ethics committee of the faculty of medicine osijek. statistical analysis categorical data are represented by absolute and relative frequencies. numerical data are described by the median and the limits of the interquartile range. category variables differences were tested by the hi-square test and, if necessary, by the fisher’s exact test. seemedj 2020, vol 4, no. 2 thyroid disease in pediatric population 80 southeastern european medical journal, 2020; 4(2) the normality of the distribution of numerical variables was tested by the shapiro-wilk test. differences of the numerical variables between the two independent groups were tested with the mann-whitney u test. all p-values are twosided. the significance level was set to alpha = 0.05. medcalc statistical software version 18.2.1 (medcalc software bvba, ostend, belgium; http://www.medcalc.org; 2018) and spss (ibm corp. released 2013. ibm spss statistics for windows) were used for statistical analysis (version 21.0. armonk, ny: ibm corp.). results categorical data are represented by absolute and relative frequencies. numerical data are described by the median and the limits of the interquartile range. category variables differences were tested by the hi-square test and, if necessary, by the fisher’s exact test. the normality of the distribution of numerical variables was tested by the shapiro-wilk test. differences of the numerical variables between the two independent groups were tested with the mann-whitney u test. all p-values are twosided. the significance level was set to alpha = 0.05. medcalc statistical software version 18.2.1 (medcalc software bvba, ostend, belgium; http://www.medcalc.org; 2018) and spss (ibm corp. released 2013. ibm spss statistics for windows) were used for statistical analysis (version 21.0. armonk, ny: ibm corp.). table 1. incidence of thyroid diseases by diagnosis during 2010 and 2017 2010 2017 newly diagnose d incidence 95% c.i newly diagnose d incidenc e 95% c.i p autoimmune thyroiditis 9 11.65 5-33 88 113.91 91-26 <0.001 congenital hypothyroidism 1 1.29 0-3 2 2.59 0-31 >0.999 unspecified hypothyroidism 4 5.18 1-41 39 50.48 35-88 <0.001 hyperthyroidism 1 1.29 0-3 1 1.29 1-41 >0.999 non-toxic goiter 15 19.42 1087 45 58.25 42-46 <0.001 other thyroid diseases 0 0.00 0-0 18 23.30 13-81 <0.001 *incidence per 100,000 inhabitants, c.i. – confidence interval table 1 presents the incidence of thyroid diseases by diagnosis during 2010 and 2017 the number of newly diagnosed cases of autoimmune thyroiditis in 2010 was 9, accounting for the incidence of 11.65 per 100,000 inhabitants aged 0-14, and in 2017, the number of newly diagnosed cases was 88, accounting for the incidence of 113 newly diagnosed children per 100,000 inhabitants. such a 9.7-fold increase in incidence was found to be statistically significant (p <0.001). also, there was a 9.75-fold increase in the incidence of unspecified hypothyroidism during the years mentioned. in 2010, the incidence of unspecified hypothyroidism was 5.18, and in 2017, 50.48 per 100,000 inhabitants (p <0.001). a threefold increase in the incidence of non-toxic goiter was detected, with an incidence of 19.42 newly diagnosed children in 2010, compared to 58.25 newly diagnosed children per 100,000 inhabitants aged 0-14 (p <0.001). other unspecified thyroid diseases are also on a statistically significant rise – no cases were reported in 2010, and in 2017, the incidence was 23.3 new cases per 100,000 inhabitants aged 014. seemedj 2020, vol 4, no. 2 thyroid disease in pediatric population 81 southeastern european medical journal, 2020; 4(2) table 2 presents distribution of patients in regard of nutritional status. there were no significant differences in relation to malnutrition, or normal nutrition and overweight/obesity among subjects examined in 2010 and 2017. table 2. distribution of subjects in relation to nutrition status number (%) of subjects p* nutrition 2010 2017 overall malnourished 3 (5) 4 (2) 7 (3) normal weight 36 (60) 117 (56) 153 (57) 0.20 overweight 12 (20) 33 (16) 45 (17) obese 9 (15) 54 (26) 63 (24) overall 60 (100) 208 (100) 268 (100) *hi-square test also, no significant differences were observed between two populations studied in terms of specific aspects of physical examination (i.e. characteristics of the skin, eyes, presence of palpitations, appetite, body mass and stool and urination, table 3). table 3. subjects according to specific aspects of physical examination number (%) children p* 2010 2017 total skin regular moisture and warmth, without trembling 67 (93) 186 (94) 253 (94) 0.77 thyroid symptomatology 5 (7) 11 (6) 16 (6) total 72 (100) 197 (100) 269 (100) eyes normal 69 (99) 196 (100) 265 (99.6) 0.26 pathology 1 (1) 0 1 (0.4) total 70 (100) 196 (100) 266 (100) palpation regular 46 (68) 132 (61) 178 (63) 0.39 enlarged 22 (32) 83 (39) 105 (37) total 68 (100) 215 (100) 283 (100) appetite regular 68 (94) 151 (92) 219 (92) 0.39 changed 4 (6) 14 (8) 18 (8) total 72 (100) 165 (100) 237 (100) body mass constant 71 (99) 161 (98) 232 (98) >0.99 fluctuating 1 (1) 4 (2) 5 (2) total 72 (100) 165 (100) 237 (100) stool regular 59 (89.4) 159 (94.6) 217 (93.1) >0.99 irregular 7 (10.6) 9 (5.4) 11 (4.7) total 66 (100) 167 (100) 233 (100) urination regular 72 (100) 165 (100) 237 (100) *fisher’s exact test seemedj 2020, vol 4, no. 2 thyroid disease in pediatric population 82 southeastern european medical journal, 2020; 4(2) presence of family history of thyroid diseases was significantly more frequent in data collected in 2017; however, no difference was observed in terms of appearance in paternal, maternal or kin side (table 4). also, comorbidities were equally distributed between two studied groups of patients (table 5). table 4. subjects by family history and years observed number (%) children p* 2010 2017 total family history positive 23 (44) 107 (67) 130 (61) 0.004† negative 29 (56) 53 (33) 82 (39) total 52 (100) 160 (100) 212 (100) positive family history maternal side 16 (73) 64 (65) 80 (61) 0.06 paternal side 2 (9) 19 (19) 21 (16) combined (brother/sister/maternal/paternal) 4 (18) 15 (16) 19 (23) total 23 (100) 107 (100) 130 (100) *fisher’s exact test; †χ2 test table 5. distribution according to comorbidities regarding the years observed number (%) of subjects p* 2010 2017 total obesity 1 (3.6) 22 (12.5) 23 (11.27) 0.213 polycystic ovary syndrome/ dysmenorrhea 0 (0) 4 (2.3) 4 (1.96) >0.999 haematological irregularities (↓trc, anaemia) 0 (0) 4 (2.3) 4 (1.96) >0.999 skin disorders (acne, psoriasis, vitiligo, atopy) 0 (0) 7 (4) 7 (3.43) 0.597 respiratory diseases of allergic etiology (allerg.rhinitis/bronchitis/asthma) 1 (3.6) 4 (2.3) 5 (2.45) 0.526 jra, polyarthralgia, sy raynaud 1 (3.6) 6 (3.4) 7 (3.43) >0.999 benign nodus 0 (0) 6 (3.4) 6 (2.94) >0.999 hypertension, heartbeat irregularities/palpitations 0 (0) 8 (4.5) 8 (3.92) 0.602 celiac disease 0 (0) 5 (2.8) 5 (2.45) >0.999 gastritis, gerb 0 (0) 2 (1.1) 2 (0.98) >0.999 trembling, vertigo, headache 1 (3.6) 11 (6.3) 12 (5.88) >0.999 epilepsy and epileptic convulsions 1 (3.6) 1 (0.6) 2 (0.98) 0.256 chromosomopathies (sy down, sy turner, sy digeorge) 0 (0) 5 (2.8) 5 (2.45) >0.999 hypotrophy, retarded growth 0 (0) 6 (3.4) 6 (2.94) >0.999 accelerated growth, pubertas praecox 2 (7.1) 4 (2.3) 6 (2.94) 0.192 hyperlipidaemia 1 (3.6) 5 (2.8) 6 (2.94) 0.593 *fisher’s exact test seemedj 2020, vol 4, no. 2 thyroid disease in pediatric population 83 southeastern european medical journal, 2020; 4(2) discussion based on the data of the total population in the republic of croatia, the incidence of newly diagnosed children with thyroid disease was estimated. since no newly diagnosed subjects were over 14 years of age (specifically, in both years, the oldest newly diagnosed subject was 12 years old), the data from the population register for ages 0-14 were used (24). due to the peculiarity of the pediatric population of eastern croatia, patients from osijek-baranja and vukovar-srijem counties gravitate mostly to the university hospital centre osijek. for this reason, the 2010 census data for osijek-baranja county and vukovar-srijem county were used to approximate the incidence of newly diagnosed subjects, which totalled to 77,257 children aged 0-14 (osijek-baranja: 46,806 children, vukovar-srijem: 30,451 children). due to the dynamics of population change and the inability to present current population values, the approximation of the population incidence was expressed at 95% confidence intervals. our results suggest that the total number of newly diagnosed cases of thyroid disease has increased significantly. a similar trend in the incidence of thyroid disease has been reported in other studies (25). looking at the incidence of thyroid disease by years, there is a statistically significant increase in the incidence of autoimmune thyroiditis, unspecified hypothyroidism, non-toxic goiter, and other unspecified thyroid diseases. the increase in the incidence of thyroid disease, especially autoimmune thyroid disease, is a global trend, present especially in western countries, where the decrease of infectious diseases incidence is also being observed (11, 26). this research confirms that a similar trend can be observed in subjects. furthermore, probably mostly due to the existence of screening for congenital hypothyroidism during the early neonatal period, figures regarding this diagnosis have not changed significantly over the years, and fortunately remained low. so far, numerous studies on gender and autoimmunity have concluded that thyroid diseases, such as autoimmune thyroiditis and goiter, are more common in women, as confirmed by this research. this pattern has been partially elucidated by the influence of female sex hormones and by x inactivation, but remains to be fully explained (27). in present study, subjects were analyzed regarding the nutrition status expressed by body mass index. patients with chromosomopathies (down syndrome, digeorge syndrome, turner syndrome) were excluded, since their growth and development do not follow the usual patterns. subjects were classified into 4 groups depending on the centile malnourished, normal weight, overweight and obese subjects. the study showed that the majority of subjects in both observed years had normal body weight (57%), 3% were malnourished, while 41% of subjects were overweight and obese. in both observed years, a large proportion of childre n were overweight and obese. in a 2010 study conducted on healthy croatian school-age children, it was found that 69.9% of children were normal weight and 30.1% were overweight and obese (28). taking into account the results of our study on the population of children suffering from thyroid disease, it is obvious that there is a slightly higher proportion of overweight and obese children (by 10.9%) among children with thyroid disorders than the population of healthy children, while the share of children with normal body weight was slightly lower (12.9%). although statistically insignificant, it could suggest a higher incidence of thyroid disease among children of a higher body mass index, as explained by the interaction of metabolically active adipose tissue and leptin and the thyroidstimulating hormone (29). specifically, adipose tissue plays a role in the pathophysiology of thyroid disorders. fat cells produce leptin, which is why they are considered to be an active endocrine organ. leptin is a potential link between the tsh and body mass index; it acts as a neuroendocrine regulator of the hypothalamus-pituitary-thyroid axis by regulating tsh gene expression in certain brain regions. vice versa, the tsh stimulates leptin secretion in adipose tissue. leptin also promotes the conversion of t4 to t3 (30). in obese children, elevated tsh and t4 levels have become a common finding. since these values appear to seemedj 2020, vol 4, no. 2 thyroid disease in pediatric population 84 southeastern european medical journal, 2020; 4(2) result from an elevated body mass index, it might be possible to normalize thyroid hormone levels by correcting or losing extra body weight. however, the incidence of thyroid antibodies is low in these patients, suggesting that adiposity via the previously explained association with leptin affects tsh levels, but not the thyroid antibody levels (31, 32). obesity has become a growing public health problem. at the same time, it also appears to be a perpetuating factor in the increased incidence of thyroid disease. other physical status data were also taken from the subjects' medical records to determine if there were significant changes between these variables in 2010 and 2017. by processing the data collected, it was concluded that there was no statistically significant difference between the variables observed in the years compared by which we could explain the increased incidence of thyroid disease. in addition to all other information, one of the characteristics of thyroid disease is certainly family clustering and a positive family history. a positive family history of thyroid disease in 2010 was present in 44% of the subjects, while in 2017, this number increased statistically significantly to 67%. given the invariability of the genetic component of the disease, the increase in the number of subjects with a positive family history could be explained by the greater awareness of patients with thyroid disease and cognition of similar diseases within their own families, but also by the indirectly increased incidence of thyroid disease in both the adult and pediatric populations (26). the most common comorbidities include obesity, diabetes mellitus type 1, cystic ovarian syndrome and dysmenorrhea, skin changes, respiratory diseases of allergic etiology, hypertension, palpitations, celiac disease, gastritis and gerd, chromosomopathies, praecox puberty and accelerated growth, which, according to available literature, have been previously linked to thyroid disease and thyroid hormone imbalance (33-42). however, the difference in the incidence of these comorbidities in the observed years was not found to be statistically significant. such persistent co-occurence might suggest that there is a pathophysiological link between some of these diseases and autoimmune thyroid disease. based on the research conducted and the analysis of the data obtained, several conclusions can be drawn. the incidence of thyroid disease in general, autoimmune thyroiditis, unspecified hypothyroidism and nontoxic goiter increased multiple times in the observed period. other unspecified thyroid diseases are also on the statistically significant rise. during the observed period, there was a statistically significant increase in the number of diseased female children compared to the male children in each year separately, but the ratio of the diseased did not change significantly during the observed years. also, there were no significant differences in comorbidities in the subjects compared to the years observed, which could explain the increased incidence. the consistency of a positive family history in the direction of thyroid disease was confirmed, with the prevalence of a positive family history along the maternal line. conclusion in conclusion, it is evident that autoimmune diseases in general are a local and global issue that will appear even more frequently in the future. with the aim of successful treatment and potential prevention, additional efforts are needed to determine the exact incidence and to identify potential risk factors for the development of these diseases. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. seemedj 2020, vol 4, no. 2 thyroid disease in pediatric population 85 southeastern european medical journal, 2020; 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139:738-740. i i author contribution. acquisition of data: tomic a, puseljic s, tomac v administrative, technical or logistic support: tomic a, puseljic s, tomac v analysis and interpretation of data: tomic a, puseljic s, tomac v conception and design: tomic a, puseljic s, tomac v critical revision of the article for important intellectual content: tomic a, puseljic s, tomac v drafting of the article: tomic a, puseljic s, tomac v final approval of the article: tomic a, puseljic s, tomac v guarantor of the study: tomic a, puseljic s, tomac v provision of study materials or patients: tomic a, puseljic s, tomac v statistical expertise: tomic a, puseljic s, tomac v seemedj 2021, vol 5, no. 1 maternal environmental factors and gastroenteritis 37 southeastern european medical journal, 2021; 5(1) original article maternal environmental factors as predictors of occurrence of gastroenteritis among under-five children in akure south local government area, ondo state 1 oluwaseyi oye olofintuyi *1, b o ogundele 2, olasunkanmi rowland adeleke *1, joseph sunday adegboro 1, rachael seun oluwadare 1 1 department of human kinetics and health education, adekunle ajasin university, akungba-akoko, nigeria 2 department of human kinetics and health education, university of ibadan, nigeria *corresponding authors: olofintuyi oluwaseyi o and adeleke olasunkanmi r, olasunkanmi.adeleke@aaua.edu.ng, oluwaseyiolofintuyi406@gmail.com received: aug 26, 2020; revised version accepted: jan 25 2021; published: apr 28, 2021 keywords: gastroenteritis, environment, mothers, infection abstract aim: to examine maternal environmental factors as predictors of the incidence of gastroenteritis among under-five children in akure south local government area, ondo state. materials and methods: a descriptive study was carried out in two state-owned hospitals between april and august 2019 using a purposive and convenience sample of 120 mothers of under-five children. data collected were analyzed using ppmc and regression to test the hypotheses at 0.05 alpha level. results: the study shows that three out of four environmental predictors were potent predictors of the incidence of gastroenteritis. they include: quality of water source (β = .387, t = 7.638, p < 0.05), method of sewage disposal (β = .508, t = 9.651, p < 0.05) and hygienic practices (β = .341, t = -6.799, p < 0.05), while area of residence (β = -.048, t = 1.008, p > 0.05) was not a potent predictor. area of residence, quality of water source, hygienic practices and method of sewage disposal had a significant joint contribution used to predict the incidence of gastroenteritis. conclusion: area of residence, quality of water source, hygienic practices and method of sewage disposal all increase the incidence of gastroenteritis when proper attention is not paid to them. education about handwashing is necessary for mothers and environmental health workers in collaboration with the ministry of health, and the ministry of environment should ensure that every house has the proper means to dispose of sewage, especially a septic tank (flush toilet); this will help reduce the disposal of feces in the environment. (olofintuyi oo, ogundele bo, adeleke or, adegboro js, oluwadare rs. maternal environmental factors as predictors of occurrence of gastroenteritis among under-five children in akure south local government area, ondo state. seemedj 2021; 5(1); 37-46) seemedj 2021, vol 5, no. 1 maternal environmental factors and gastroenteritis 38 southeastern european medical journal, 2021; 5(1) introduction children’s health is central to the growth and development of any nation’s economy, as they are the future leaders. a child’s health needs to be addressed immediately, from the day when the mother learns she has conceived. therefore, it is imperative that the mother provides adequate support to a child’s emotional, mental and physical well-being by helping them make healthy choices about hygienic and sanitation practices. over the years, babies and children below the age of five experience one health challenge or another due to a variety of factors, which may be hereditary, genetic, congenital or environmental in nature, and which often lead to admission into hospitals and an alarming rate of infant mortality. gastroenteritis, also known as infectious diarrhea, is the irritation of the gastrointestinal tract – the stomach and the small intestine. it is often accompanied by abdominal pains, diarrhea and vomiting (1). gastroenteritis can affect people of all age groups (both adults and children can be affected). persons who are most at risk are individuals with low immunity, especially infants and elderly adults. according to the world health organization (who), over 70% of diarrhea‐related deaths among children less than 5 years old occur in africa (2), and nigeria has one of the highest under‐5 mortality rates in the world (328 deaths per 100 000) (3). recent studies have shown an increase in the prevalence of rotavirus-induced diarrhea in different parts of nigeria. for instance, a study conducted in benin city (4) reported a prevalence rate of 19.2%, and one in sokoto, northern nigeria, a high prevalence rate of 25% (5). in ibadan (6), a prevalence of 18.5% was reported, while kaduna recorded a prevalence rate of rotavirus-induced gastroenteritis of 32.2% among under-five children (7). moreover, a study (8= conducted in akure recorded a high percentage of incidence (31%) in children in the age group between 0 and 12 months. the incidence of gastroenteritis remains a significant burden on children in developing countries due to a range of elements, such as the lack of available healthcare services, lack of safe drinking water, poor sanitation, poor hygiene of both child and caregiver and overcrowding (1). the transmission of infectious diarrhea (gastroenteritis) can be related to the area of residence. the risk of contracting gastroenteritis can increase as a result of animals living with humans or in close proximity to human dwellings; they can transmit it directly to humans or through contaminated water and overcrowding (9). any airborne and respiratory infection is likely to spread in overcrowded areas, especially among infants because of their weak immune system. the quality of drinking water is also linked with the likelihood of suffering from a gastroenteritis infection. the source of water fit for drinking should be considered in regard to its nearness to any toilet facility, in order to avoid water contamination. several reference works have revealed that children with access to safe drinking water are less likely to suffer from gastroenteritis than those who use water from unsafe sources (10, 11). likewise, poor handling and storage of drinking water is seen to be significantly associated with an increased risk of infectious diarrhea (12-14). the method of sewage disposal by the caregiver in a household is quite significant for the incidence of gastroenteritis among under-five children. no access to a hygienic toilet may lead to large amounts of fecal waste being discharged into the environment, especially without adequate treatment, which is likely to have a major impact on infectious disease burden and quality of life (15(. the prevalence of diarrhea is lower among children who live in a house with less dirty sewage than in children who do not (16). poor food hygiene practices have been reported to increase the risk of diarrhea episodes among infants when unsterilized feeding utensils and bottle feeders are used (17). the mothers’ practice of handwashing before food preparation is associated with a lower risk of diarrhea among children. there is a high risk of seemedj 2021, vol 5, no. 1 maternal environmental factors and gastroenteritis 39 southeastern european medical journal, 2021; 5(1) diarrhea among children aged < 2 years if the mother has poor food hygiene practices (18). some findings (19, 20) also reveal that a combination of environmental factors has an effect on the rapid increase of gastroenteritis. the role of a mother cannot be overemphasized as most activities that help prevent gastroenteritis take place in the home, and the outcome of the illness depends greatly on the initial steps or actions taken by the mothers at home. due to the high incidence of gastroenteritis in children under five years of age in akure south local government area, ondo state, compared to the incidence rate in other parts of nigeria, this study aimed to investigate the maternal environmental factors as predictors of the incidence of gastroenteritis among under-five children in akure south local government area of ondo state. we hypothesized that: 1) there is no significant relationship between the area of residence, quality of water source, hygienic practices, method of sewage disposal and the incidence of gastroenteritis among under-five children in akure south local government area of ondo state; 2) there is no significant relative contribution of maternal environmental factors (area of residence, quality of water source, hygienic practices, method of sewage disposal) on the incidence of gastroenteritis among under-five children in akure south local government area of ondo state; and finally, 3) there is no significant joint contribution of maternal environmental factors (area of residence, quality of water source, hygienic practices, method of sewage disposal) to the incidence of gastroenteritis among under-five children in akure south local government area of ondo state. materials and methods a descriptive research design of the correlational type was used for this study. this study was conducted between april and august 2019. the population for this study comprised of mothers of under-five children in akure south local government area of ondo state. the sample used for this study included one hundred and twenty (120) mothers of under-five children selected from the mother and child hospital, akure, and ondo state general hospital in akure south local government area of ondo state. purposive sampling technique was used to select two (2) state-owned hospitals in akure south local government area of ondo state; both are dedicated to the care of pregnant mothers and children, have a high level of patronage and are located in densely populated areas. convenience sampling technique was used to select a number of mothers whose children under the age of five have experienced or are currently affected by the infection. the instrument used for this study was a selfdeveloped questionnaire designed by the researchers in line with the variables under study. the instrument was validated by making a draft copy available for criticism to experts in the field of maternal and health education, which led to subtraction, addition and possible modification of the research instrument. the corrected version of the instrument was used for the collection of data. it was validated by three experts in the field of health education and its reliability was ensured by using cronbach alpha with the coefficient value of 0.79 obtained. the descriptive statistics of frequency counts was used to present background information of the respondents, while inferential statistics of regression was used to test the research hypotheses at the 0.05 level of significance. results table 1 shows the relationship between each independent variable (access to clean water, sanitation facilities used, hygienic practices and area of residence) and the dependent variable (incidence of gastroenteritis); there is a significant relationship between the incidence of gastroenteritis and quality of water source (r = 0.054, p < 0.05), sanitation facilities used (r = 0.739, p < 0.05) and hygienic practices (r = 0.461, p < 0.05), but not area of residence (r = 0.148, p > 0.05). hygienic practices (mean = 13.81) are the highest contributing factor to reducing the incidence of gastroenteritis among under-five children based on the mean value, followed by seemedj 2021, vol 5, no. 1 maternal environmental factors and gastroenteritis 40 southeastern european medical journal, 2021; 5(1) sanitation facilities used (mean = 12.78), while area of residence (9.68) has the lowest mean value. table 1. correlation matrix showing the relationships between study variables. variables mean std. dev 1 2 3 4 5 p-value incidence of gastroenteritis 49.7760 5.70624 1.000 access to clean water 11.1083 1.88669 .540 1.000 0.00 sanitation facilities used 12.7833 2.18160 .739 .347 1.000 0.00 hygienic practices 13.8083 1.19731 .461 -.069 .274 1.000 0.00 area of residence 9.6750 1.91044 .148 .001 .078 .178 1.000 0.106 source: field survey (2019); keys: 1 = incidence of gastroenteritis, 2 = access to clean water, 3 = sanitation facilities used, 4 = hygienic practices, 5 = area of residence (p is significant if < 0.05) table 2 shows the relative contribution of the independent variables (area of residence, access to clean water, hygienic practices and sanitation facilities used) to the prediction of incidence of gastroenteritis. the results showed the following: access to clean water (β = .387), sanitation facilities used (β = .508) and hygienic practices (β = .341), while area of residence (β = .048) is not significant.. table 2. relative effect of environmental factors on the prediction of incidence of gastroenteritis environmental factors unstandardized coefficients standardized coefficients t sig. b std. error beta (constant) -4.012 3.656 -1.097 .275 quality of water source 1.171 .153 .387 7.63 8 .000 method of sewage disposal 1.328 .138 .508 9.65 1 .000 hygienic practices 1.623 .239 .341 6.79 9 .000 area of residence .143 .142 .048 1.00 8 .316 source: field survey (2019) table 3 reveals a significant joint contribution of the independent variables (area of residence, quality of water source, hygienic practices, method of sewage disposal) to the prediction of incidence of gastroenteritis. the result yielded a coefficient of multiple regressions r = 0.865 and seemedj 2021, vol 5, no. 1 maternal environmental factors and gastroenteritis 41 southeastern european medical journal, 2021; 5(1) multiple r-square = 0.748. this suggests that the combination of four factors accounted for 74% (adj.r2 = .740) variance in the prediction of the incidence of gastroenteritis. the anova result from the regression analysis shows a significant influence of maternal environmental factors on the incidence of gastroenteritis, f (4, 115) = 85.549, p < 0.05. table 3. summary of regression for the joint contributions of environmental factors to the prediction of incidence of gastroenteritis r = .865 r square = .748 adjusted r square = .740 std. error = 2.91120 model sum of squares df mean square f sig. 1 regression 2900.143 4 725.036 85.549 .000b residual 974.635 115 8.475 total 3874.778 119 source: field survey (2019) discussion the study revealed that quality of water source has a significant relationship with the incidence of gastroenteritis. the table further reveals that a poor-quality source of water will increase the incidence of gastroenteritis. this may happen if the water used by mothers for their children comes from a bad-quality source, which may be in close proximity to where the toilet is located. children with access to safe drinking water are almost 20% less likely to suffer from gastroenteritis than those who use water from unsafe sources, such as unprotected dug wells or springs, tanker truck/cart and surface water (11). in the same vein, (10) affirmed that the probability that a child would contract diarrhea increases for households that drink from streams at a significant level of five percent (5%). thus, households that use streams as their main source of drinking water are 0.032 more likely to have children suffering from diarrhea when compared to those living in households that use piped water as their main source of drinking water. hygienic practices have a significant relationship with the incidence of gastroenteritis. the findings of this study revealed that a high percentage of mothers of under-five children always wash their hands before preparing their child’s meal as well as after their children defecate or urinate; they also reheat their child’s meal. this indicates that a good number of mothers always practice both food and hand washing hygiene. likewise, the outcomes of this study have shown that there is a positive correlation and a significant relationship between hygienic practices and the incidence of gastroenteritis among mothers of under-five children. the mothers’ practice of handwashing before food preparation was associated with a lower risk of diarrhea among children; there is also a significant association between the incidence of gastroenteritis and washing hands with soap after cleaning the infants’ perineum (18). infants whose mothers sometimes or never wash their hands with soap after cleaning the infant’s perineum were more likely to have diarrhea than infants whose mothers always wash their hands with soap after cleaning their infant’s perineum. that is to say, the practice of handwashing reduces the risk of gastroenteritis. the type of sanitation facilities used for sewage disposal shows a significant relationship with the seemedj 2021, vol 5, no. 1 maternal environmental factors and gastroenteritis 42 southeastern european medical journal, 2021; 5(1) incidence of gastroenteritis in this study. the disposal practices of fecal waste of the youngest children were significantly correlated with the prevalence of gastroenteritis. children whose mothers reported disposing of the fecal waste of their youngest child in a pit toilet/latrine reported the highest prevalence of diarrhea in comparison to those who throw it into the garbage or rinse it away. thus, children who lived in a house with less dirty sewage had a significantly lower risk of having gastroenteritis than children who did not (16). this finding is further confirmed by the submission (15) that the proportions of children from households that use a latrine or diaper for stool disposal suffer from diarrhea less when compared to those that do not. the type of sanitation facility used showed that parents and children living in houses with non-flush toilets are twice as likely to suffer from diarrhea compared to houses with a flush toilet. children living in households with a separate flush toilet are about 50% less likely to suffer from diarrhea than children with access to a pit latrine, dry toilet and toilet shared by other households; they are generally unhygienic and pose a higher risk for the children to get gastroenteritis. regarding the relationship between the area of residence (where the mothers live) and the incidence of gastroenteritis, the study has shown that there is no significant relationship. the observation from the results of this study revealed that a significant portion of the respondents reside in a dirty environment, a place surrounded by stagnant water which could be a breeding place for flies, and even that domesticated animals also reside with them. this indicates that the environment where the mothers reside may be a place for infection to be comfortably transmitted. this is in line with the affirmation of (9) that households in rural areas with domesticated animals in close proximity show an increased risk of gastroenteritis. the presence of animals in close proximity to human dwellings adds to the risk of transmission of zoonotic infections to humans directly or through contaminated water. this study also revealed that when the environmental factors such as hygienic practices, sanitation and quality of water source are combined together, they have a significant relationship with gastroenteritis. this is confirmed from the findings of (19), where in a 2week study conducted regarding the prevalence of diarrhea, they discovered that it was 23.1%, and that residence, availability of latrine, availability of a handwashing facility, source of water, and waste disposal practices were independently associated with diarrhea. furthermore, the findings of (20) showed that many factors, such as socio-economic, behavioral like breastfeeding and environmental factors such as water, sanitation and method of waste disposal are linked to gastroenteritis. environmental factors such as the type of water source, presence of sanitation facilities, solid waste disposal system and floor type in the kitchen are found to be crucial contributors to the high prevalence of diarrhea and gastroenteritis. conclusion based on the results of this study, it was concluded that: 1) gastroenteritis is a major health problem that has over the years assumed greater significance in developing countries like nigeria; 2) many maternal factors emanating from the environment, such as area of residence, method of sewage disposal, quality of water source and hygienic practices were assessed and most were found to be significant predictors of the incidence of gastroenteritis, except for area of residence; and 3) however, the conglomeration of maternal environmental factors has a significant relationship with the incidence of gastroenteritis. recommendations at this point, it is recommended that environmental health workers in collaboration with the ministry of health and the ministry of environment ensure that every house has proper means of disposing sewage, especially a septic tank (flush toilet), as this will help reduce the disposal of feces in the environment. likewise, the government and other well-to-do seemedj 2021, vol 5, no. 1 maternal environmental factors and gastroenteritis 43 southeastern european medical journal, 2021; 5(1) people in the society should work hand in hand to provide potable water supply, which the people can easily access, as well as a way to maintain it. health educators should also train mothers on handwashing hygiene procedures and show them simple ways of how it can be done for them to apply it over time. mothers should be encouraged to practice handwashing as it prevents infection. water and sanitation interventions in urban slums should be highly effective in combating the incidence of this disease among children. acknowledgement. we appreciate prof. ogundele b.o. and prof. ayenigbara g.o. for their time to proofread this paper and make corrections where necessary. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. 1. schlossberg d. clinical infectious disease (second ed.); 2015. p. 334. isbn 978-1-107-038912. 2. world health organization. children: reducing mortality fact sheet. http://www.who.int/mediacentre/factsheets/f s178/en/index.html.2013. 3. gbd 2015 mortality and causes of death collaborators. global, regional, and national life expectancy, all‐cause and cause‐specific mortality for249 causes of death, 1980–2015: a systematic analysis for the globalburden of disease study 2015. lancet. 2016; 388:1459‐1544. 4. iyoha o, abiodun po. human rotavirus genotypes causing acute watery diarrhea among under-five children in benin city, nigeria. nigerian j clin pract. 2015; 18(1): 48–51. 5. alkali br, daneji ai, magaji aa, bilbis ls. clinical symptoms of human rotavirus infection observed in children in sokoto, nigeria. adv virol. 2015;2015:890957. doi: 10.1155/2015/890957 6. omatola ca, olusola ba, odiabo gnn. rotavirus infection among under five children presenting with gastroenteritis in ibadan, nigeria. arch basic appl med. 2016; 4(1):44-49. 7. mohammed a, aminu m, ado s, jatau e, esona m. prevalence of rotavirus among children under five years of age with diarrhea in kaduna state, nigeria. nigerian j paediatrics. 2016; 43(4):264-268. 8. kattula d, francis mr, kulinkina a, sakar r, mohan vr, babji s, ward hd, kang g, balraj v, naumova en. environmental predictors of diarrhoeal infection for rural and urban communities in south india in children and adults. epidemiol infec. 2015; 143(14):3036-47. doi: 10.1017/s0950268814003562. 9. okebugwu qc, adebolu tt, ojo ba. incidence of rotavirus infection in children with gastroenteritis in akure, ondo state, nigeria. futa journal of research in sciences. 2017; 13(1):122-128. 10. gloria e, abigail p, john aa. water and sanitation as predictors of child diarrhoea in ghana. asian journal of economics, business and accounting. 2017; 3(1):1-13. 11. aditya s, mahesh ns. diarrhea and acute respiratory infections among under-five children in slums: evidence from india. peer journal pre prints 2014. 12. oloruntoba, sridhar mk. bacteriological quality of drinking water from source to household in ibadan, nigeria. afr j med medical sci. 2007; 36(2):169–175. 13. trevett af, carter rc, tyrrel sf. water quality deterioration: study of household drinking water quality in rural honduras. int j environ health res. 2004;14(4): 273–283. 14. jinadu mk, olusi so, agun ji, fabiyi ak. childhood diarrhea in rural nigeria: studies on prevalence, mortality and socio-environmental factors. j diarrheal dis res. 1991;9(4): 323–327. seemedj 2021, vol 5, no. 1 maternal environmental factors and gastroenteritis 44 southeastern european medical journal, 2021; 5(1) 15. oloruntoba eo, folarin tb, ayede ai. hygiene and sanitation risk factors of diarrhoeal disease among under-five children in ibadan, nigeria. afr health sci. 2014; 14(4):1001–1011. 16. desmennu at, oluwasanu mm, johnakinola, yo, oladunni o, adebowale as. maternal education and diarrhea among children aged 0-24 months in nigeria. afr j repr health. 2017; 21[3]: 27-36. 17. dairo md, ibrahim tf, salawu at. prevalence and determinants of diarrhea among infants in selected primary health centres in kaduna north local government area, nigeria. pan afr med j 2017; 28:109. doi: 10.11604/pamj.2017.28.109.8152. 18. ogbo fa, agho k, ogeleka p, woolfenden s, page a, eastwood j. global child health research interest group. infant feeding practices and diarrhea in sub-saharan african countries with high diarrhea mortality. plos one 2017;12(2)e0171792. 19. workie gy, akalu ty, barak ag. environmental factors affecting childhood diarrheal disease among under-five children in jamma district, south wello zone, northeast ethiopia. bmc infect dis. 2019;19:804. 20. getachew a, alebachew t, mulat gh, tadesse g, daniel h, zemichael g, marta a. environmental factors of diarrhea prevalence among under five children in rural area of north gondar zone, ethiopia. ital j pediatr. 2018; 44:95 https://doi.org/10.1186/s13052-018-0540-7 . seemedj 2021, vol 5, no. 1 maternal environmental factors and gastroenteritis 45 southeastern european medical journal, 2021; 5(1) questionnaire on maternal environmental factors as predictor of occurrence of gastroenteritis among under-five children in akure south government area, ondo state instruction: please tick (√) the most appropriate column that suits your answer background information 1. age (in years): (a) 15-20 [ ] (b) 21-25 [ ] (c) 26-30 [ ] (d) 31-35 [ ] (e) 36-40 [ ] (f) 41-45 [ ] (g) 46 and above [ ] 2. educational status: (a) none [ ] (b) primary [ ] (c) secondary [ ] (d) tertiary [ ] 3. family size (in person): (a) 1-5 [ ] (b) 6 and above [ ] 4. monthly income (in naira): (a) ≤ 15000 [ ] (b) 16000-25000 [ ] (c) 26000-35000 [ ] (d) 36000-45000 [ ] (e) 4600060000 [ ] (f) 61000-10000 [ ] (g) 101000-150000 (h) 151-250000 [ ] (i) above 250000 [ ] section b instruction: please tick (√) the most appropriate column to indicate the extent to which you agree or disagree with the statements below. s/n quality of water source always sometimes not at all 1. open well 2. bore hole 3. river or stream 4. bottled water to drink 5. treated water method of sewage disposal always sometimes not at all 6. septic tank 7. pit toilet 8. open field defecation 9. river 10. in a bucket hygienic practices always sometimes not at all 11. i do not wash my hands before preparing my child’s food after feeding my child 12. i do wash my child’s hands after defecating or urination 13. i reheat food before my child eats 14. i wash my child’s feeding bottle 15. i wash my hands before and after i clean up my child’s feces area of residence yes maybe no 16. i live in a crowded environment 17. animals live around my house 18. my environment is free from flies 19. i reside in a place where water stores 20. i live in a clean and tidy environment seemedj 2021, vol 5, no. 1 maternal environmental factors and gastroenteritis 46 southeastern european medical journal, 2021; 5(1) occurrence of gastroenteritis infection once twic e more than twice never 21. i have a child who has suffered from this illness before 22. it affects all my children when they are between the ages of two and five 23. it comes when i stop breastfeeding my child 24. it affects my child when taken to day care center 25. it starts with my child in the community 26. it has affected the child of my neighbor before transfer to my child 27. it has affected my child even when i get immunization for the child 28. it has affected my child who did not take immunization 29. it affects my male child 30. it affects my female child 31. how many times does your child vomit in one day? 32. what is the frequency of diarrhea in one day? author contribution. acquisition of data: olofintuyi oo, ogundele bo, adeleke or, adegboro js, oluwadare rs. administrative, technical or logistic support: olofintuyi oo, ogundele bo, adeleke or, adegboro js, oluwadare rs analysis and interpretation of data: olofintuyi oo, ogundele bo, adeleke or, adegboro js, oluwadare rs. conception and design: olofintuyi oo, ogundele bo, adeleke or, adegboro js, oluwadare rs. critical revision of the article for important intellectual content: olofintuyi oo, ogundele bo, adeleke or, adegboro js, oluwadare rs. drafting of the article: olofintuyi oo, ogundele bo, adeleke or, adegboro js, oluwadare rs. final approval of the article: olofintuyi oo, ogundele bo, adeleke or, adegboro js, oluwadare rs. provision of study materials or patients: olofintuyi oo, ogundele bo, adeleke or, oluwadare rs statistical expertise (statistical analysis of data): olofintuyi oo, ogundele bo, adeleke or, adegboro js seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 1 southeastern european medical journal, vol 1, 2017. implementation of the n terminal prob-type natriuretic peptide test in national guidelines for diagnosis of heart failure in croatia1 rinea barbir1, matea zoric1. sonja perkov1. darko pocanic2, tomo svagusa2, ingrid prkacin2 1 department of medical biochemistry and laboratory medicine, merkur university hospital, zagreb, croatia 2 department of internal medicine, merkur university hospital, zagreb, croatia corresponding author: rinea barbir rineabarbir@gmail.com received: march 5, 2017; revised version accepted: april 19, 2017; published: april 24. 2017 keywords: new natriuretic peptide test, laboratory precision abstract aim: our aim was to implement n terminal prob-type natriuretic peptide (nt-probnp) testing at the department of medical biochemistry and laboratory medicine (dmblm), accredited according to the hrn en iso 15189 standard, for clinical use at the emergency unit of the internal medicine department in merkur university hospital. methods: electro-chemiluminescence immunoassay (eclia) is a sandwich principle test with two monoclonal nt-probnp-specific antibodies. precicontrol cardiac ii level 1 and level 2 were analyzed using the eclia on roche cobas e411, in triplicate, for five consecutive days, for the purpose of calculating within-laboratory precision, according to the clinical and laboratory standards institute (clsi) protocol. we prospectively studied 87 emergency department (ed) patients with symptoms of decompensated heart failure (hf) during one month, measuring their nt-probnp levels. results: according to the clsi protocol, we calculated standard deviation and coefficient of variation for repeatability, intermediate precision and within-laboratory precision from control results. calculated coefficient of variation for the overall laboratory precision for level 1 and level 2 was within the desirable biological criteria for precision, and within the manufacturer’s criteria for overall laboratory precision. we assessed the association between the new nt-probnp method and the outcome in hf patients during one month, and showed the distribution of nt-probnp values in our ed patients. conclusion: results indicate that the nt-probnp test met all the set criteria and it has been implemented at the dmblm for clinical use in merkur university hospital, according to croatian national guidelines for diagnosis of heart failure. (barbir r, zoric m, perkov s, pocanic d, svagusa t, prkacin, i. implementation of the n terminal prob-type natriuretic peptide test in national guidelines for diagnosis of heart failure in croatia. seemedj 2017;1(1);1-4) seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 2 southeastern european medical journal, vol 1, 2017. introduction croatian guidelines have been prepared in accordance with the new 2016 european society of cardiology (esc) guidelines (1) and they note the use of n terminal prob-type natriuretic peptide (nt-probnp) biomarker as the first line of diagnosis of heart failure (hf), which enables rapid diagnosis and proper cardiac monitoring. the guidelines were launched on november 3, 2016, the first day of the 11th congress of the croatian cardiac society. the guidelines were accepted with great enthusiasm by the participants – both primary care physicians and cardiology specialists. our aim was to implement the nt-probnp test at the department of medical biochemistry and laboratory medicine (dmblm), accredited according to the hrn en iso 15189 standard, for clinical use at the emergency unit of the internal medicine department in merkur university hospital. materials and methods eclia is a sandwich principle test with two monoclonal nt-probnp-specific antibodies. in the first incubation antigen in the sample, a biotinylated monoclonal nt-probnp-specific antibody and a monoclonal nt-probnp-specific antibody, labeled with ruthenium complex, form a sandwich complex. after addition of streptavidin-coated microparticles, the complex becomes bound to the solid phase via interaction of biotin and streptavidin. the reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. after addition, tripropylamine voltage is applied to the electrode, which induces chemiluminescent emission (2). accreditation requirements include the need to verify the method performance prior to using it on patient samples (3). during verification, lyophilized control samples were stored at 2-8°c and stabilized at 20-25°c before measurement. precicontrol cardiac ii level 1 and level 2 with values of 150 ng/l and 4930 ng/l were analyzed by the eclia on roche cobas e411, in triplicate, for five consecutive days, in order to calculate within-laboratory precision, according to the clinical and laboratory standards institute (clsi) protocol: user verification of performance for precision and trueness, ep15-a2 (4). according to the protocol, we calculated arithmetic mean and standard deviation for each day of measurement, from which we determined the repeatability for each day, as well as the standard deviation for intermediate precision for all five days. standard deviation for within-laboratory precision was calculated from the square root of the sum of standard deviation for repeatability and intermediate precision. we prospectively studied the emergency department (ed) patients with symptoms of decompensated heart failure (hf) during one month, and measured their nt-probnp levels. we included 87 consecutive patients in whom hf was determined based on clinical symptoms and congestive hf symptoms were determined based on admission chest x-ray. patients without decompensated heart failure were excluded. patients’ characteristics were: females/males = 41/47 (45%/55%), age: females 77 (range 52 – 92 years) and males: 65 (range 44 – 86 years). the study was approved by the local ethics committee and written informed consents to participate were obtained. results we implemented the nt-probnp test according to the clsi protocol and calculated standard deviation (sd) and coefficient of variation (cv) for repeatability, intermediate precision and withinlaboratory precision from the control results. cv calculated for within-laboratory precision was 4,48% for level 1 and 4,15% for level 2 (table 1).. calculated coefficients of variation for withinlaboratory precision for two control samples were compared with coefficients of variation for the two set criteria, desirable biological criteria for precision and manufacturer’s criteria for within-laboratory precision (table 2). seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 3 southeastern european medical journal, vol 1, 2017. from january 5 to february 9, the concentration of nt-probnp was measured in 87 patients, with repeated measurements for 18 patients. 20 – 30% increase in concentration was observed in three patients (3/87, 1f/2m) in the period of ten days after admission, with a fatal end (short-term mortality in 1 month). the highest nt-probnp value was detected in a man with cardiac shock (>70000n/l). results of the nt-probnp levels are shown in figure 1. discussion despite very good curative cardiology, croatia is still among the countries with high cardiovascular risk and mortality. therefore, croatian guidelines for hf diagnosis were developed according to the esc guidelines. ntpro bnp is a proven diagnostic and prognostic biomarker for acute and chronic hf. it is highly recommended by the esc guidelines (1). natriuretic peptides predict cardiovascular events in patients and are associated with prognosis in decompensated heart failure (5). implementation of the nt-probnp test was conducted according to the clsi protocol – calculated cv for the within-laboratory precision was 4,48% for level 1 and 4,15% for level 2, which was compared with the set criteria. desirable biological criteria for precision was 5,00%, according to ricos c. and colleagues (6), and manufacturer’s criteria for within-laboratory precision was also 5,00 %, which indicates that table 1. calculated standard deviation (sd) and coefficient of variation (cv) for repeatability, intermediate precision and withinlaboratory precision. (n – number of measurements per day, d – number of days) control samples (n = 3, d = 5) repeatability intermediate precision within-laboratory precision preci control cardiac ii sd (ng/l) cv (%) sd (ng/l) cv (%) sd (ng/l) cv (%) level 1 (149 ng/l) 4,04 2,66 5,97 3,92 6,82 4,48 level 2 (4920 ng/l) 83,96 1,77 189,98 3,89 197,27 4,15 table 2. achieved coefficients of variation for within-laboratory precision and set criteria preci control cardiac ii manufacturers criteria for the overall laboratory precision achieved withinlaboratory precision desirable biological criteria for precision cv (%) cv (%) precision 0,5 cvintra (%) level 1 (149 ng/l) 5,00 4,48 5,00 level 2 (4920 ng/l) 5,00 4,15 5,00 figure 1. distribution of 105 nt-probnp values (min. 8,29 ng/l-max. 70000 ng/l) seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 4 southeastern european medical journal, vol 1, 2017. the achieved results were within the set criteria, desirable biological criteria for precision and manufacturer’s criteria for within-laboratory precision, respectively (7). concentration of ntprobnp in samples obtained from the ed patients indicate good negative predictive value of the test, whereas high values were correlated with a higher mortality risk (8). in our paper, elevated nt-probnp values were strongly predictive of adverse outcomes and rising values identified a rising risk, but lowering of ntprobnp denoted improved outcomes. thus, the direction of change is important. very high nt-probnp concentration is an independent predictor of adverse outcomes in patients (9). serial nt-probnp testing provides very important clinical information. monitoring of hf with nt-probnp serial testing provides more clinical information than single testing (1). implementation of the nt-probnp test could lower the number of repetitive referrals to ed, help improve the quality of service and reduce unnecessary waste of resources. conclusion results indicate that the nt-probnp test met all the set criteria and it has been implemented at the dmblm since january 5, 2017, for clinical use at the emergency unit of the internal medicine department in merkur university hospital, in accordance with croatian national guidelines for diagnosis of hf. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. ponikowski p, voors aa, anker sd, bueno h, cleland jg, coats aj, falk v, gonzálezjuanatey jr, harjola vp, jankowska ea, jessup m, linde c, nihoyannopoulos p, parissis jt, pieske b, riley jp, rosano gm, ruilope lm, ruschitzka f, rutten fh, van der meer p; authors/task force members. esc guidelines for the diagnosis and treatment of acute and chronic heart failure. european heart journal. 2016; 37: 2129-2200. 2. ecl technology, roche diagnostics elecsys 2010 immunoassay system – reference guide v4-1. 3. zlata flegar-meštrić, aida nazor, sonja perkov, branka šurina, mirjana mariana kardum-paro, zoran šiftar, mirjana sikirica, ivica sokolić, ivan ožvald, željko vidas. accreditation of medical laboratories in croatia. coll antropol. 2010; 34: 181-186. 4. clinical and laboratory standards institute – user verification of performance for precision and trueness; approved guideline – second edition, ep15-a2, 2006. 5. scrutinio d, ammirati e, guida p, passantino a, raimondo r, guida v, sarzi braga s, pedretti rf, lagioia r, frigerio m, catanzaro r, oliva f. clinical utility of n-terminal pro-b-type natriuretic peptide for risk stratification of patients with acute decompensated heart failure. derivation and validation of the adhf/nt-probnp risk score. int j cardiol. 2013; 168(3): 2120-2126. 6. ricos c, alvarez v, cava f, garcia-lario jv. current databases on biologic variation: pros, cons and progress. scand j clin lab invest. 1999; 59: 491-500. 7. ricós c, álvarez v, minchinela j, fernándezcalle p, perich c, boned b, gonzález e, simón m, díaz-garzón j, garcía-lario jv, cava f, fernández-fernández p, corte z, biosca c. biologic variation approach to daily laboratory. clin lab med. 2017; 37(1): 47-56. 8. huang yt, tseng yt, chu tw. n-terminal pro b-type natriuretic peptide (nt-pro-bnp)based score can predict in-hospital mortality in patients with heart failure. scientific reports. 2016; 6: 29590. pmid 27411951. doi: 10.1038/srep29590. 9. santaguida pl, don-wauchope ac, ali u, oremus m, brown ja, bustamam a, hill sa, booth ra, sohel n, mckelvie r, balion c, raina p. incremental value of natriuretic peptide measurement in acute decompensated heart failure (adhf): a systematic review. heart fail rev. 2014; 19(4):507-519. seemedj 2020, vol 4, no. 1 sleep disorders in hemodialysis treatment patients 74 southeastern european medical journal, 2020; 4(1) review article factors associated with sleep disorders in patients undergoing chronic hemodialysis treatment 1 đorđe pojatić 1, 2, davorin pezerović 2, 3, dubravka mihaljević 1, 4, dunja degmečić* 1, 5 1 faculty of medicine, josip juraj strossmayer university of osijek, croatia 2 general county hospital vinkovci, vinkovci, croatia 3 faculty of dental medicine and health, josip juraj strossmayer university of osijek, croatia 4 department of internal medicine, clinical hospital centre osijek 5 department for psychiatry, clinical hospital centre osijek *corresponding author: dunja degmečić, ddegmecic@gmail.com received: jan 14, 2020; revised version accepted: feb 17, 2020; published: apr 27, 2020 keywords: hemodialysis, depression, sleep disorder abstract introduction: patients undergoing chronic hemodialysis treatment (hd patients) experience sleep quality deterioration, which is associated with lower quality of life and represents an independent predictor of mortality in hd patients. recently, the number of research papers aimed at assessing sleep quality in hd patients has been increased, due to the fact that it is such an important factor. thus, this study aimed to identify the main factors related with low sleep quality in hd patients by reviewing scientific literature. materials and methods: a search based on key words was performed in the medline database and selected scientific papers were analyzed by using the matrix method. the initial search retrieved 472 scientific articles to which certain inclusion and exclusion criteria were applied, combined with a critical analysis, resulting in the selection of a total of 48 papers. factors related with low sleep quality in hd patients were grouped in three sub-groups, while sociodemographic and clinical characteristics showed significant impact on low sleep quality in hd patients. results: it has also been proven that low sleep quality is correlated with biochemical variables such as inflammation factors, low albumin levels and other factors, whereas knowledge concerning factors involved in hemodialysis also contributes to the efforts of medical staff aimed at improving hd patients' quality of sleep. conclusion: etiology of sleep quality deterioration in hd patients is manifold, and significantly affected by psychosocial, biochemical and clinical factors. (pojatić đ, pezerović d, mihaljević d, degmečić* d. factors associated with sleep disorders in patients undergoing chronic hemodialysis treatment. seemedj 2020; 4(1); 74-86) seemedj 2020, vol 4, no. 1 sleep disorders in hemodialysis treatment patients 75 southeastern european medical journal, 2020; 4(1) introduction end-stage chronic kidney disease (ckd) is characterized by glomerular filtration rate below 15 ml/min, presence of oliguria and electrolyte imbalance. end-stage ckd requires hemodialysis treatment. the hemodialysis procedure can be extremely uncomfortable and painful for patients, who tend to suffer from muscle spasms and drop of arterial blood pressure. in addition, from a long-term perspective, patients with this disease have a lower quality of life due to high incidence of depressive disorder, sleep disorder, secondary alexithymia and other factors related to hemodialysis, comorbidities and general health status of the patient. disturbances of sleep quality and other sleep disorders constitute a factor that significantly reduces the quality of life of this group of patients and that is associated with the incidence of depressive disorder, at the same time being an independent predictor of mortality in patients undergoing hemodialysis (hd patients) (1, 2). prevalence of sleep disorders in hd patients is 41%-83%, depending on the results of the conducted research. in addition, etiology of sleep disorders is manifold and complex (3). sleep disorders are classified according to their most important features. we thus distinguish the following sleep disorders: insomnia, breathing-related sleep disorders, parasomnia, hypersomnia, disorders of the circadian wake/sleep rhythm and disorders related to movements of extremities (4). breathing-related sleep disorders, which encompass both obstructive sleep apnea and disorders related to movements of extremities such as restless legs syndrome, are significantly more common in hd patients than in the general population; however, risk factors that contribute to the development of these disorders have not yet been fully elucidated (5, 6). due to disturbances in endogenous melatonin production cycle and the intermittent character of hemodialysis, hd patients tend to suffer more from disturbances of the circadian wake/sleep rhythm (7). in their studies on sleep disorders in hd patients, majority of researchers decided to focus on sleep quality considering that it is a comprehensive and widely accepted concept and that implies both quantitative and qualitative aspects of sleeping (8). sleep quality is a concept that encompasses duration of sleep, latency before falling asleep, sleep efficiency and the patient’s subjective assessment of whether they feel refreshed after sleep. for this reason, the majority of authors use instruments for assessment of the quality of sleep in their studies (8). the objective of this paper was to conduct a review of medical literature and determine the impact of socio-demographic, clinical, laboratoryand hemodialysis-related factors on deteriorated quality of sleep in chronic hemodialysis patients. methods the searched scientific database was medline and the search was performed by using the key words „hd patients“ and „sleep quality“. studies that did not contain these key words in their title or in the abstract were excluded from the search. literature search included original scientific papers, qualitative research and case reports, whereas results of scientific reviews or letters to the editor were not the subject of analysis. most of the articles were published over the course of last ten years, including some dating back even earlier than that, which were included due to the fact that they provided significant knowledge. this resulted in the analyzed period being the period from 1999 to 2019. articles that focused on the pediatric population and therapeutic procedures for treatment of sleep disorders unrelated to hemodialysis (except new knowledge about melatonin), as well as articles that were not published in english, were excluded from the analysis, as were articles focused on analyzing the quality of sleep in transplantees and peritoneal dialysis patients. seemedj 2020, vol 4, no. 1 sleep disorders in hemodialysis treatment patients 76 southeastern european medical journal, 2020; 4(1) results database search based on the abovementioned key words and the analyzed time period retrieved 472 scientific articles. after application of the mentioned elimination criteria and exclusion of scientific reviews, analysis was reduced to 93 articles. based on a detailed examination of the titles and abstracts of the articles against the objectives of this scientific review, 48 articles were identified as suitable, 17 of which were free-access articles, whereas 31 articles required access authorization. final analysis included 48 studies published in the period from 1999 to 2019, 35 of which were cross-sectional, 4 were prospective studies, 8 were randomized clinical trials and one was a qualitative study. results of the studies listed provide insights into the recent discoveries regarding correlation between sleep quality in hd patients and laboratory variables, levels of depression and alexithymia, factors related with hemodialysis, as well as regarding the impact of specific sleep disorders, such as restless legs syndrome, on sleep quality in hd patients (table 1).. table 1. factors associated with low sleep quality in hd patients table 1. factors associated with low sleep quality in hd patients a) psychosocial factors alexithymia it implies difficulty distinguishing between feelings and bodily sensations; in hd patients, it predicts low sleep quality independently of depression. depression depression reduces sleep quality; prevalence of depression among hd patients is four times higher compared to general population. b) sociodemographic factors female gender, old age, low physical activity, high body mass index these factors are related to decreased sleep quality in hd patients independently of depression. poor financial status, unemployment poor financial status and unemployment impair sleep quality. c) biochemical factors crp, il-6, tnf-alfa, il1beta sleep disorders in hd patients are associated with high levels of proinflammatory and low levels of antiinflammatory cytokines. decreased appetite, low serum albumin and creatinine levels researchers assess nutritional parameters together with inflammation using the malnutrition inflammation score, which is an independent predictor of low sleep quality in hd patients. secondary hyperparathyroidism, hyperphosphatemia high pth, phosphate and calcium levels impair sleep quality in hd patients. loss of circadian rhythm of melatonin secretion hd patients have a disrupted melatonin rhythm; administration of exogenous melatonin improves sleep quality. d ) factors related to hemodialysis procedure dialysis shift and relative hyperhydration high relative hyperhydration causes sleep disorders in hd patients; patients treated in the nocturnal shift have better sleep quality. presence of tunneled central venous catheter these hd patients more often have muscular cramps and uremic pruritus, which causes low sleep quality. seemedj 2020, vol 4, no. 1 sleep disorders in hemodialysis treatment patients 77 southeastern european medical journal, 2020; 4(1) assessment of sleep disorders in hd patients was performed by using the pittsburgh sleep quality index (psqi), epworth sleepiness scale (ess), the berlin questionnaire and the stop bang questionnaire for measuring obstructive sleep apnea, rls and irlssg questionnaires for assessment of restless legs syndrome severity, insomnia severity scale (iss), cheq (choice health experience questionnaire), holland sleep disorders questionnaire, hatoum's sleep questionnaire, and athens insomnia scale (ais).there were two studies that used polysomnography and two that used actigraphy. three of the studies analyzed different sleep variables by using individual questions, which represents a limitation of those studies. as far as assessment of depressive symptoms is concerned, researchers used the beck depression inventory (bdi-i and ii), the center for epidemiologic studies depression scale (cesd), the hospital anxiety and depression scale (has-d), the taiwanese depression questionnaire (tdq), and the patient health questionnaire (phq-2). for assessment of alexithymia, the toronto alexithymia scale-20 (tas-20) was used. the final result of this integrative scientific literature review was the formation of three subtopics in this context, one being recent discoveries in the correlation between depression, quality of life and quality of sleep and the other two being laboratory variables and hemodialysis-related variables.. discussion correlation between depression, alexithymia, socio-demographic factors and sleep disorders in hd patients alexithymia is a construct, a stable personality trait that implies difficulty distinguishing between feelings and bodily sensations, difficulty describing feelings to other people, constricted imaginal processes and a cognitive style of thinking that is oriented towards external stimuli rather than feelings (9). prevalence of alexithymia is higher in hd patients than in the general population, as high as 30%, this being a cause of their significantly reduced life quality (1). alexithymia is a strong predictor of mortality in hd patients, regardless of age, sex or possible depressive disorders. patients suffering from alexithymia had a 4.29 % higher mortality rate at later stages of the five-year monitoring period (1). the way alexithymia affects sleep disorders has not been sufficiently elucidated; there have only been a few studies dealing with this issue on samples of hd patients and it has long been unclear whether the impact of alexithymia on sleep quality deterioration was indirect or direct. gennaro et al. conducted a large cross-sectional study on a population of students and reached a conclusion that alexithymia was a confounding factor and that if depression is included in the statistical models, its impact on the quality of sleep disappears (10). researchers that analyzed the same issue on a sample of hd patients confirmed the impact of alexithymia on poorer quality of sleep, but on a sample of hd patients, this impact was not correlated with depression. in particular, de santo et al. used a sample of 40 hd patients who underwent parathyroidectomy due to high pth levels and who were found to suffer from high intensity depression, alexithymia and sleep disorders. their study showed that the impact of alexithymia on sleep disorders was not related with levels of depression. however, the study was limited by a small number of subjects, which represents a weakness inherent in the study (11). although research into the correlation of alexithymia and biochemical variables in hd patients is scarce, it has been confirmed that alexithymia, or rather its component in the form of external stimulioriented cognitive thinking, represents an independent predictor of low phosphorus levels in hd patients, which are in turn related with poor quality of sleep (12). depressive disorder is a mood disorder defined as a feeling of sadness, unhappiness, loss of will and motivation that lasts for more than two weeks. it is one of the most common mental disorders, whereas sleep disturbance is one of the most prominent symptoms of depressive disorder (13). based on available discoveries, seemedj 2020, vol 4, no. 1 sleep disorders in hemodialysis treatment patients 78 southeastern european medical journal, 2020; 4(1) prevalence of depression in hd patients is almost four times higher than in the general population and amounts to between 43% and 72% (14). depression has a significant impact on sleep disturbances in patients receiving chronic hemodialysis treatment, it considerably reduces the quality of life and is an independent predictor of patients’ mortality (1, 15, 16). social support has a preventive effect when it comes to depressive disorder and improves the quality of life of hd patients (17). according to the study carried out by pan et al. , the form of social support that includes analytical appraisal of virtues and positive personality traits of such patients reduces the impact of disturbed sleep quality on life quality and depression in hd patients (17). in their cross-sectional study, maung et al. found that depressive hd patients tend to suffer more frequently from daytime sleepiness (measured using ess), lower sleep quality, restless legs syndrome as well as from sleep-related difficulties the night after receiving hemodialysis treatment (15). latest discoveries point to the importance of additional factors that modify this two-way interaction. according to the results of recent research, sleep disturbances are significantly more frequent in women than in men receiving chronic hemodialysis treatment (1820). only one study found higher incidence of low quality of sleep in the male sex, but the study was conducted on a sample of malaysian subjects, so it is possible that such result was caused by culturological and racial differences (19). scientists have also considered the role of depression as a confounding factor in this relationship because it is more frequent in women; however, recent studies have identified female sex to be an independent risk factor when it comes to development of sleep disorders during hemodialysis treatment (14, 21). it is interesting to note that according to the psqi, the age of hd patients also represents an independent risk factor for sleep disorder. however, lin et al. found that this influence disappears if monthly pecuniary income of respondents is taken into account. it is possible, as the authors indicated, that the impact of age was dependent on financial status, as financial status drops significantly in the population of retirees, so this is something that should definitely be taken into consideration in future studies (21). financial status and unemployment were defined in two studies as the factors that contribute to poor sleep quality in hd patients (23, 24). tomita et al. found that duration of hemodialysis treatment could also result in poor sleep quality measured by the psqi questionnaire. specifically, patients who have been undergoing dialysis for more than five years exhibit more difficulty in functioning during the day as a result of daytime sleepiness, which is an element measured by the psqi questionnaire, whereas independent analysis shows the correlation between advanced age and sleep disorders, regardless of the levels of patients' depression (24). lower physical activity of hd patients has an independent negative effect on the quality of sleep. namely, using a large sample of 1678 patients undergoing peritoneal dialysis and hemodialysis covered by cds (comprehensive dialysis study), anand et al. found that lower physical activity is more frequent in depressive patients, but also that independent of depression, low physical activity functions as a factor that causes poor sleep quality (25). apart from lower physical activity, studies have shown that bmi (body mass index) is another factor that negatively affects the quality of sleep in hd patients (27, 28). it is a well-known fact that depression has a seasonal character and that depressive symptoms in patients suffering from depressive disorder as well as depression-related insomnia or hypersomnia tend to become worse during certain seasons. according to the results of afsar et al., seasonal remission of depression symptoms is not accompanied by an improved quality of sleep. authors claim that remission of depression symptoms can be perceived only in non-smoking patients and in those with increased levels of serum albumin during the summer (28). many research studies have been conducted on the impact of laboratory variables on sleep disorders and depression in hd patients and seemedj 2020, vol 4, no. 1 sleep disorders in hemodialysis treatment patients 79 southeastern european medical journal, 2020; 4(1) most of them concluded that laboratory variables have a stronger impact on depression than on sleep disorders, which is logical since depression is a disorder caused by biochemical imbalance of neurotransmitters, whereas sleeping can be affected by many different socio-economic factors (14). afsar et al. established that aggravation of chronic kidney disease is accompanied by higher cortisol levels and that these increased cortisol levels in turn cause higher depression frequency. however, this factor does not cause sleep disorders (29). depression in hd patients is connected with elevated levels of phosphorous, low levels of creatinine and hemoglobin, and low albumin levels, which serve as indicators of their nutritional status (21, 22, 30) . when discussing disturbances of sleep quality and depression in patients, we must not disregard religious feelings and beliefs, which have been identified, in the context of general population, as factors that help patients to overcome stress even when other anti-stress mechanisms falter. eslami et al. proved that a high level of spiritual well-being and religious feelings have an independent and positive effect on sleep quality in hd patients (30). effect of biochemical variables on sleep disorder in hd patients measured using psqi when talking about recent discoveries, one needs to mention the correlation between sleep disorders and inflammatory mediators, which has been confirmed by results of many studies. the factor that all of these studies focus on is the relatively nonspecific but highly sensitive creactive protein (crp), the level of which many cross-sectional studies found to be significantly higher in hd patients suffering from sleep disorders (28, 32). the study conducted by chiu et al. reveals that the level of pro-inflammatory cytokine il-1beta, which is responsible for triggering inflammatory responses in the human body, is significantly increased in patients who are undergoing chronic hemodialysis treatment and who are suffering from sleep disorder, and that there is a correlation between the levels of other pro-inflammatory cytokines such as tnfalpha or il-6 and deteriorated sleep quality (32). it is possible that elevated pro-inflammatory mediators are the consequence rather than the cause of sleep disorder because, as specified by chiu et al., sleep deprivation may cause a temporary increase in arterial blood pressure, which then triggers inflammatory response and the synthesis of crp in the liver cells (32). results obtained by the group of researchers led by taraz confirm the pro-inflammatory theory, albeit with reference to lower levels of antiinflammatory cytokine il-10 in patients suffering from sleep disorder; however, the importance of this study is limited by the small number of respondents (33). it is possible that lower vitamin d level found in hd patients is the cause of higher levels of pro-inflammatory cytokines tnf, il-6 and il-1 as well as lower sleep quality; nevertheless, it is also possible that there is an independent mechanism by which vitamin d affects vitamin d receptors in areas of the brain regulating sleep (34). conversely, crp levels represent a more trustworthy proof of the importance of inflammation when it comes to sleep disturbances, because crp levels are stable during the 24-hour period, whereas cytokine secretion reaches its maximum level according to its specific circadian rhythm (34). the study carried out by razeghi and ali included 108 hd patients and revealed that patients who had crp levels above 3.8 ug/l were more prone to suffer from poor sleep quality according to the pittsburgh sleep quality index (psqi), including insomnia, restless leg syndrome (rls) and obstructive sleep apnea syndrome (35). however, it is necessary to emphasize that several studies did not reveal any kind of correlation between crp and proinflammatory cytokine levels on the one side and poor sleep quality on the other (36). another factor that is closely related to inflammatory components and their indirect or direct impact on sleep quality is the nutritional status of patients suffering from end-stage chronic kidney disease. (37). the patient’s nutritional status and loss of appetite are closely related to dietary restrictions for hd patients, mental condition, age as well as to the level of seemedj 2020, vol 4, no. 1 sleep disorders in hemodialysis treatment patients 80 southeastern european medical journal, 2020; 4(1) inflammatory components. for this reason, some researchers assess these parameters together using malnutrition inflammation score (mis) (31). mis and albumin level represent independent predictors of poor sleep quality in hd patients. research focusing on albumin serum levels obtained the most reliable results, which almost always point to inverse correlation between albumin levels and sleep quality (28, 35, 39). correlation between low albumin levels on the one side and shorter sleep duration and lower sleep efficiency one the other has also been proven by polysomnography (psg), which is a gold standard in sleep disorder research (39). dilek ongan found that, according to their scale, patients with moderate appetite were exposed to 3.26-fold higher risk of poor sleep quality, whereas patients with bad appetite were exposed to 4.2-fold higher risk of developing sleep disorders (40). an additional confirmation that nutritional status is associated with sleep quality is strong correlation between creatinine levels and sleep quality in the examined groups of patients, considering that the serum creatinine level is a consequence of good appetite. in addition, dilek ongan revealed a positive effect that intake of vitamins b1, b6 and b9 has on sleep quality (40). only a very small number of studies concluded that a patient’s nutritional status has no impact on the issue in question (34, 37). previous studies found that metabolic acidosis and venous bicarbonate level are associated with higher frequency of obstructive sleep apnea episodes in samples of population not suffering from chronic kidney disease, whereas results of the study conducted on a sample of hd patients confirmed an independent impact of venous bicarbonate and ph levels on poor sleep quality measured by psqi (38). restless legs syndrome (rls), which is significantly more common in hd patients, is an independent predictor of sleep disturbances, whereas its occurrence is also correlated with higher levels of crp and white blood cells (41). patients with rls have an uncontrollable urge to move their lower extremities due to an uncomfortable feeling in their legs. these symptoms occur during the night when patients are motionless. rls is not caused by other pathological conditions such as diabetes. the uncomfortable feeling is experienced before the person has moved his or her legs (42). it has been found that homocysteine may potentially be the cause of rls, considering that the levels of this amino acid are higher in rls positive patients, provided that the portion of homocysteine bound to serum albumin is taken into account (43). loss of circadian rhythm of melatonin secretion is an abnormality detected in hd patients, which clearly has an impact on sleep quality; however, the impact-related mechanisms have not yet been thoroughly examined. application of melatonin therapy in a randomized controlled clinical trial carried out by nejad et al. proved that exogenous melatonin administration had a positive impact on sleep quality, sleep efficiency and contributed to lower disturbances during sleep in hd patients with previous sleep disorders (44). it is well-known that application of melatonin during twelve months causes resistance to treatment, lower sleep quality, impaired endogenous melatonin secretion and loss of circadian rhythm of secretion (45). it has also been observed that application of melatonin in a study conducted by nejad et al. reduced the quantity of erythropoietin required to correct the hemoglobin value, which was also correlated in some studies with poorer quality of sleep in hd patients (46). as early as in 1999, peterson found, by polysomnography, reduced periodic limb movements and sleep fragmentation as well as total sleep prolongation and improved daytime alertness in a sample of patients suffering from chronic kidney disease whose anemia was successfully corrected by application of short-acting erythropoietin (47). according to more recent studies, the impact of anemia on sleep quality is questionable. there are many studies that failed to prove the correlation between lower hemoglobin levels and sleep quality, which is most likely the result of the fact that this group of studies did not take into account application of erythropoietin as a confounding factor (18, 32). that such studies are nevertheless carried out is justified by the fact that some researchers have seemedj 2020, vol 4, no. 1 sleep disorders in hemodialysis treatment patients 81 southeastern european medical journal, 2020; 4(1) identified anemia as an independent cause of nocturnal sleep fragmentation and daytime sleepiness in hd patients (33, 35). secondary hyperparathyroidism is a consequence of end-stage chronic kidney disease because the body uses higher levels of parathyroid hormone (pth) in order to adapt to the initial hypocalcemia, which is the result of lower vitamin d synthesis and hyperphosphatemia (48). hyperparathyroidism in end-stage chronic kidney disease is accompanied by high levels of calcium and phosphorous. it seems that pth is the cause of sleep disorders, depression and alexithymia in patients only when the value of pth is particularly high and cannot be treated by pharmacological therapy (11). de santo et al. proved lower pth levels and ameliorated depression, alexithymia and sleep disorders after parathyroidectomy, after having studied a sample of 40 hd patients (49). studies that examined pth levels using a sample of patients receiving pharmacological therapy obtained contradictory results when it comes to the effect of pth levels on sleep quality. namely, nejad et al. identified significantly lower levels of intact pth in the group of patients with sleep disorders, whereas taraz et al. identified significantly higher levels (34, 51). patients suffering from restless legs syndrome, which has already been identified as a predictor of sleep disturbances, also have higher levels of parathyroid hormone (43). pth has a direct impact on brain tissue. in a study in which magnetic resonance imaging was used, ma et al. found a decreased n-acetylaspartate to creatine ratio in the thalamus of hd patients, which is significantly associated with the level of parathyroid hormone and patients’ psqi score (51). n-acetylaspartate is the marker of axonal stability in brain tissue and its decreased ratio in comparison with the ratio found in patients not suffering from chronic kidney disease is a sign of axonal injury. discoveries concerning the impact of ionized phosphorous are unique and the impact of calcium levels on sleep quality deterioration remains unclear. hyperphosphatemia is a clear factor and predictor of low quality of sleep (20, 35, 37). impact of factors related to characteristics of hemodialysis treatment on deteriorated sleep quality the fact that hemodialysis shift may impact sleep quality and circadian rhythm alteration was examined in several studies, with conflicting and contradictory results. in their study that included 220 respondents, wang et al. successfully proved that better sleep quality is associated with morning-shift dialysis and with already familiar sleep disturbance factors such as depression and anxiety (52). the authors made a critical remark regarding the results of their study, stating that they did not take into account napping during dialysis as a possible confounder and that nocturnal sleep quality potentially depends on this variable. however, firoz and hosseini did not prove either that there is a correlation between hemodialysis shift and deteriorated sleep quality or that sleep quality is affected by change of the dialysis shift (23). undergoing nocturnal hemodialysis has certain advantages in terms of sleep duration being better in hd patients undergoing hemodialysis in the night shift than in the case of hemodialysis in two daytime shifts. furthermore, the intensified hemodialysis program, within which hemodialysis is performed six times per week, is associated with better sleep quality. this correlation does not, however, show statistical significance (36). the quality of hemodialysis treatment is defined by the dose of hemodialysis required by patients with end-stage chronic kidney disease in order to ensure that their mortality and life quality parameters are the same as those of patients not suffering from chronic kidney disease (54). it is clinically defined as urea clearance passing through the dialyzer multiplied by the duration of an individual hemodialysis session, divided by urea distribution volume in the body (54). urea distribution volume is total water volume in the body less ultrafiltration volume in an individual hemodialysis session. recent studies have not succeeded to indicate a correlation between the quality of hemodialysis and deteriorated sleep quality (23, 56). in their study, unruh et al. divided patients into daily and nocturnal hemodialysis shifts during a one-year period and at the end of seemedj 2020, vol 4, no. 1 sleep disorders in hemodialysis treatment patients 82 southeastern european medical journal, 2020; 4(1) this period they found sleep quality to be higher in the nocturnal group, which group also had better hemodialysis quality. however, the results were not statistically significant (54, 57). one study proved a lower level of disturbances of sleep quality in patients who have a hemodialysis arteriovenous fistula (avf) in comparison with patients who undergo hemodialysis using tunneled central venous catheter (55). according to the results obtained by hilda et al., patients who undergo hemodialysis via an arteriovenous fistula are less prone to suffer from muscular cramps and uremic pruritus, which conditions are associated with poor sleep quality (55). uremic pruritus or itch occurs as a result of higher levels of phosphorus and uremic toxins, and it is also caused by lower hemodialysis quality (2, 56). during hemodialysis treatment, the level of ultrafiltration or the volume of fluid that is removed from the patient’s body represents a special challenge. bioimpedance is the standard for estimation of dry body weight, which in turn determines the level of ultrafiltration. studies in which the said method was used in order to study the impact of fluid overload and low ultrafiltration on the quality of sleep revealed predominantly consistent results. relative hyperhydration in hd patients, which is defined as surplus bodily fluids above 7%, is independently associated with lower sleep quality and daytime sleepiness (57). abreo et al. found that relative hyperhydration has an impact on lower sleep duration, more frequent nighttime waking and more difficulty returning back to sleep (58). studies that are focused on different forms of renal replacement therapy usually compare treatment by standard hemodialysis with hemofiltration and hemodiafiltration. as opposed to hemodialysis, in addition to the physical process of diffusion of small molecules, hemofiltration also uses conduction laws for quick flow of dialysate solution, which enables removal of larger harmful molecules (54). hemodiafiltration is a combination of these two methods. in their cross-sectional study, hilda et al. found that hemodiafiltration has a positive impact on disturbances of sleep quality only in male patients (55). on the other hand, in their prospective study, gu et al. found that at the end of a two-year period, treatment using a combination of hemodialysis and hemofiltration, which took up half of the hemodialysis treatment session (hd+hf), had a positive impact on pth, calcium, phosphorus, crp and melatonin levels, as well as on sleep quality (59). the study also indicated that hd+hf treatment of patients is associated with an increase in the overall survival in comparison with patients who are treated only by hemodialysis (59). conclusion previous research studies focusing on the topic of sleep quality in hd patients have consistently arrived at the conclusion that depression has an independent impact on sleep quality in hd patients and that alexithymia has an indirect and significant impact on sleep quality in hd patients. depression is a factor that is often accompanied by alexithymia and that reduces the quality of life of hd patients and it is an independent predictor of mortality. results obtained by studies examining the impact of biochemical variables on sleep quality are inconsistent, indicate higher level of correlation with the depressive disorder and fail to provide an answer regarding various confounding factors such as anemia medications, electrolyte imbalance and pth levels. future studies should include a prospective approach, they should use more objective methods such as polysomnography or actigraphy and take into account the impact of medications on the said biochemical variables. the results obtained by studies focused on dialysis and vascular access modalities lead to the conclusion that creation of an av fistula in hd patients and usage of hemodiafiltration method are justified because of their impact on better sleep and life quality and their contribution to lower frequency of muscle cramps after a hemodialysis session. given that sleep quality is a factor that is independently associated with life expectancy of hd patients, future studies should focus on seemedj 2020, vol 4, no. 1 sleep disorders in hemodialysis treatment patients 83 southeastern european medical journal, 2020; 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51:140-147. doi: 10.1016/j.sleep.2018.07.011 58. abreo ap, dalrymple ls, chertow gm, kaysen ga, herzog ca, johansen kl. predialysis volume overload and patientreported sleep duration and quality in patients receiving hemodialysis. hemodial int. 2016; 21(1):1–9. 59. gu yh, yang xh, pan lh. additional hemoperfusion is associated with improved overall survival and self-reported sleep disturbance in patients on hemodialysis. int j artif organs. 2019; (1):1–7. seemedj 2017, vol 1, no. 1 the dual nature of the antiepileptic drug valproic acid… 74 southeastern european medical journal, vol 1, 2017. the dual nature of the antiepileptic drug valproic acid, with possible beneficial effects in alzheimer’s disease 1 domagoj đikić1, david jutrić1, kristina dominko2 1 department of animal physiology, faculty of science, university of zagreb, 10000 zagreb, croatia 2 institute “ruđer bošković”, bijenička cesta 54, croatia corresponding author: domagoj đikić, phd magistar_djikic1@yahoo.com introduction valproic acid (vpa) is a short, branched-chain fatty acid with anticonvulsant properties and one of the most widely prescribed drugs for many received: march 15, 2017; revised version accepted: april 24, 2017; published: april 24. 2017 keywords: hepatotoxicity, metabolic disorders, lipid transport, steroidogenesis, alzheimer’s disease types of epilepsy in children and adults. typically, it is applied as the sodium salt form, the sodium valproate and vpa, or mixture of both compounds. the therapeutic concentration varies between 50 and 150mg/l in the serum (1abstract valproic acid (vpa) is a short fatty acid with strong anticonvulsant properties. it has diverse effects in different tissues with opposing mechanisms of physiological action. due to the effects on energy, fatty acid, and cholesterol metabolism, it may be a risk factor for the development of diabetes with its associated complications of atherosclerosis, weight gain, hypertension, insulin resistance and other complications. its negative effects on the endocrine system can have severe health consequences, especially in the female population. vpa produces proinflammatory and proapoptotic effects in the liver and anti-inflammatory and antiapoptotic effects in the central nervous system. it also causes abnormalities in lipid and cholesterol transport in the liver and the reproductive organs, while in neural stem cells it decreases cholesterol accumulation and helps neural growth and differentiation. however, in the cns it has some beneficial effects, which are proposed to be important in alzheimer’s disease (ad). in ad mouse models, vpa exerted antiapoptotic effects and the expression of transcription factors that promote neurite growth. most of the adverse pathogenic actions or beneficial molecular effects are not fully understood. we present an overview and comparison of the different properties of vpa and their effects on estrogen and cholesterol metabolism, lipid transport, alzheimer’s disease, and on the physiology of the liver, reproductive organs, and neurons from in vitro and in vivo (in animal models and patients) studies. (đikić d, jutrić d, dominko k. the dual nature of the antiepileptic drug valproic acid, with possible beneficial effects in alzheimer’s disease. seemedj 2017;1(1);74-89) seemedj 2017, vol 1, no. 1 the dual nature of the antiepileptic drug valproic acid… 75 southeastern european medical journal, vol 1, 2017. 3). the lc-ms/ms or chemiluminescent microparticle immunoassay are used for determination of the vpa level in plasma (4). at serum concentrations 200mg/l and above, vpa can have adverse drug reactions, and it is necessary to lower the dosage of the drug to avoid the acute overdose toxic effects that may include hyperthermia/hypothermia, tachycardia, hypotension (with severe overdose), serious respiratory depression necessitating airway assistance, and cardiac arrest (with severe overdose). the central nervous system (cns) findings in cases of vpa overdose may include: coma, confusion, somnolence, worsened seizure control, dizziness, hallucinations, irritability, headache, ataxia, and cerebral edema. death by overdose and death associated with chronic complications in adults are recorded as well as death among children (1, 5). although vpa is very efficient in treating epilepsy, its chronic therapeutic application is associated with many unwanted adverse drug reactions in certain prone patients (6).adverse drug reactions, which are described within this manuscript from cited literature sources, include: weight gain, atherosclerosis, hypertension, nonalcoholic fatty liver disease (nafld), alterations of adipocytokine homeostasis, increased oxidative stress, insulin resistance, severe liver toxicity, death, abnormalities in estrogen and androgen metabolism, and pcos (polycystic ovary syndrome) (figure 1). on the other hand, experimental data, which are described in manuscripts from cited literature sources, suggest that vpa also has effects which seem to be beneficial regarding the physiology of neural tissue (figure 2), especially in alzheimer’s disease. the effects of vpa in the cns seem to be completely opposite of those occurring in the liver and other tissues. by survey of the literature in table 1, major search engines yield between 15000 and 17000 hits when using the key word valproate. in the last 6 years, there have been 2862 (scopus) and 4087 (pubmed) published papers on the subject of vpa (table 1). this review has been written to summarize the latest evidence of vpa’s effects in living organisms. novel physiological roles are being discovered, which we point to in the last chapter of the review. metabolism, physiological and molecular effects absorption, distribution, metabolism and elimination (adme) of valproic acid pharmacokinetic and pharmacodynamic studies show that the absorption of short fatty acid vpa in the gastrointestinal system is close to 100 % (7,8). because the molecular structure of vpa is similar to other short and long chain fatty acids, it follows the absorption pathways of all other triglycerides, cholesterol, and fatty acids.nearly 90% of vpa is bound to the serum proteins. only the unbound portion of the compound is active.the biological halftime of elimination in patients is 6-16 hours (1).the time of elimination is extended in children younger than 18 months of age. in the organism, there are at least 50 table 1. the results of literature survey on two search engines using the key word valproate. search engine total no. of reference s (19712017) year 2017 (first trimester) year 2016 year 2015 year 2014 year 2013 year 2012 scopus 15050 86 437 519 596 598 626 pubmed 17146 203 782 747 805 788 762 seemedj 2017, vol 1, no. 1 the dual nature of the antiepileptic drug valproic acid… 76 southeastern european medical journal, vol 1, 2017. known metabolites. the vpa molecule is partially metabolized in the cytoplasm, and partially in the mitochondrion where it is transported by the help of carnitine. within the cytoplasm, it is oxidized in phase i reactions by cytochrome p-450(cyp450)-catalyzed oxidation with the major enzymes being cyp2a6, cyp2b6 and cyp2c9, which form major toxic metabolites 4-enevpa and 2-ene vpa. cytoplasmic vpa can be immediately conjugated to udp-glucuronic acid by udp-glycotransferases (ugt1a3, -1a4, 1a6, -1a7, -1a8, -1a9, -1a10 and -1a15). the 4ene vpa and vpa molecules enter the mitochondrion conjugated to coa (4-enevpacoa and vpa-coa) where they undergo mitochondrion-mediated β-oxidation and cytochrome p-450(cyp450)-catalyzed oxidation. the major metabolites of cyp450catalyzed oxidation are the 2-propyl-4pentenoic acid (4-ene-vpa), and the β-oxidation metabolite 2-propyl-2, 4-pentadienoic acid (2, 4diene-vpa). these metabolites are considered to be the main hepatotoxic metabolites of vpa. the metabolite 2-propyl-2-pentenoic acid (2ene-vpa), which forms through the mitochondrial β-oxidation, is also converted to the 2, 4-diene-vpa in vivo (1;7;9). in phase ii of the biotransformation reactions, the majority of the formed metabolites are subjected to further glucuronidation, which is considered the major route for vpa and metabolite elimination. to a lesser extent, the adverse metabolite (2,4-dienevpa-s-coa) can be conjugated with glutathione (gsh) to form thiol conjugates, but more importantly it causes a decrease in mitochondrial glutathione levels, as described in rat hepatocyte cell cultures (9). the vpa glucuronides and their glucuronidated metabolites are excreted primarily by the urinary route and only in a small amount through the bile and into the intestines (1;7; 10). figure 1. an overview of the physiological and adverse drug reaction effects caused by valproic acid and sodium valproate treatment. seemedj 2017, vol 1, no. 1 the dual nature of the antiepileptic drug valproic acid… 77 southeastern european medical journal, vol 1, 2017. mechanism of therapeutic action it is thought that the major physiologic mechanism of vpa’s therapeutic properties in epilepsy is the inhibition of 4-aminobutyrate aminotransferase (abat), a transaminase in the gamma aminobutyric acid (gaba) pathway. therefore, vpa increases gaba release. it may also act by altering the properties of voltagedependent sodium channels. in this way, it stops a seizure attack during epilepsy. vpa is also a histone deacetylase inhibitor. with this function, it promotes more transcriptionally active chromatin structures, which is a likely epigenetic mechanism for the regulation of many of the neuroprotective effects attributed to valproic acid. intermediate molecules mediating these effects include the vascular endothelial growth factor (vegf), the brain-derived neurotrophic factor (bdnf) and the glial cell line-derived neurotrophic factor (gdnf) (1, 10, 11). adverse drug reactions liver adverse effects in the liver vpa causes an accumulation of lipids, cholesterol, and fatty acids with the subsequent development of hepatic steatosis and nonalcoholic fatty liver disease (nafld) in patients as well as in in vitro and in vivo models.the hepatic adverse drug reactions caused by steatosis are probably due to the abnormalities in lipid transport, but the exact mechanisms of liver adverse drug reactions are not clear. neither are the mechanisms by which vpa induces nonalcoholic fatty liver disease (nafld). experimentally on human hepg2 cell lines, in vivo, vpa treatment causes leakages of figure 2. an overview of the physiological effects caused by valproic acid and sodium valproate in neural tissue. seemedj 2017, vol 1, no. 1 the dual nature of the antiepileptic drug valproic acid… 78 southeastern european medical journal, vol 1, 2017. alt, ast, and ldh in a dose dependent manner suggesting injury to hepatocytes (12). the 4-ene-vpa and 2, 4-diene-vpa metabolites and enzymes cyp1a1, cyp2a6, cyp2c9, abcg1, and cpt1a are associated with hepatic adverse drug reactions in humans and in certain hepatic cell lines (8;12). however, only the cyp2a6 polymorphism was found to be associated with higher concentrations of 4-ene-vpa and 2, 4diene-vpa. potential important risk factors include mutated genotypes of cyp2c9 and cyp2a6, and higher concentrations of vpa (8). exposure to sodium vpa has been shown to induce a down regulation of several transcripts in cultured hepatocytes. their low levels resulted in time dependent fluctuations of cellular atp, which can lead to cell death (13). hepatic adverse drug reactions caused by vpahave also been associated with mitochondrial dysfunctions, with the inhibition of enzymes in the beta-oxidation pathwayand with oxidative stress.as aforementioned, vpa can form thiol conjugates particularly with glutathione (9). such decreases of intracellular glutathione levels are known to change the redox status of the cell and lead to the adverse drug reactions of oxidative stress effects. for example, vpa incubation of rat hepatocytes in vitro caused glutathione depletion which suggests a possible cellular oxidative stress after vpa exposure (9). for vpa, the role as a potential oxidant is underexplored, and there are scarce literature sources on the subject. experiments on human hepg2 cell lines showed that exposure to vpa for more than 72h increased levels of mitochondrial reactive oxygen species production (ros), but decreased protein levels of mitochondrial superoxide dismutase sod2, suggesting oxidative stress caused by impaired elimination of mitochondrial ros (13). pourahmad et al. (14) and jafarian et al. (15), both, in vitro, detected increases in ros formation along with a decrease in mitochondrial membrane potential upon the treatment of rat liver mitochondria with vpa; all of these were events before cell death signaling began. specifically, jafarian et al. (15) showed that ros is associated with increased lipid peroxidation, mitochondrial membrane collapse; mitochondrial swelling and finally the release of cytochrome c. release began apoptotic cell death signaling. similarly, pourahmadet al. (14) in their experiment demonstrated that the cytotoxic action of vpa manifests itself as lysosomal membrane leakiness in conjunction with ros formation and a decline in membrane potential.again, all were events before cell lysis started. chang et al. (16) investigated the genetic polymorphisms in the g protein beta three subunit (gnb3) and associations with the metabolic phenotypes of vpa treated human patients.their study confirmed that patients, who are t allele carriers of gnb3 c825t polymorphism, have a lower risk of vpa induced metabolic abnormalities.they warrant further studies investigating mechanisms of vpa induced metabolic abnormalities and g protein. an article by stewart et al. (17) describes that the risk of hepatic adverse drug reactions induced by vpa is increased in patients with mitochondrial diseases, especially those with polg1 gene mutations. polg genes code for the mitochondrial dna polymerase gamma and their mutations can cause alpers-huttenlocher syndrome, a neurometabolic disorder linked to an increased risk of developing fatal hepatic adverse drug reactions upon exposure to vpa. stewart et al. (17) also described an association of vpa induced hepatic adverse drug reactions with a genetic variation in the polg gene. in their case, it was a heterozygous genetic variation in the polg gene which was primarily due to the p.q1236h substitution, and it was strongly associated with vpa induced liver toxicity. in their experiment on primary human cell lines, therapeutic doses of vpa stopped human cellular proliferation and high doses of vpa induced non-apoptotic cell death, not mediated by mitochondrial dna depletion, a defect infatty acid metabolism or mutation.therefore, another mechanism of liver injury by vpa is impaired cellular liver regeneration.one of the ways of preventing it is by prospective genetic testing for polg which could identify individuals at high seemedj 2017, vol 1, no. 1 the dual nature of the antiepileptic drug valproic acid… 79 southeastern european medical journal, vol 1, 2017. risk for potentially fatal consequences of vpa treatment (17). hepatic lipid metabolism is impaired during vpa treatment. the secretion of triacylglycerols and phospholipids at the sinusoidal pole of hepatocytes are reduced by an acute administration of vpa. this inhibition of secretion has been thought by berllinger et al. (18) to be a factor in the development of steatogenic hepatic adverse drug reactions by vpa treatment (18) in rat hepatocytes, though so far shown only in an animal model. gene expression profiles in hepatocytes could be associated with the steatogenic hepatic adverse drug reactions of sodium vpa. gene profiling data showed striking changes in the expression of genes associated with lipid, fatty acid, steroid metabolism, oncogenesis, and signal transduction and development in mice treated with vpa (19). lee, et al. (19), found that 1156 genes were up and down regulated after exposure to sodium vpa. 60 genes were involved in lipid metabolism and were interconnected with the biological pathways for biosynthesis of triglycerides and cholesterol, catabolism of fatty acids and lipid transport (19). obesity, fatty acid, and cholesterol disorders in patients treated with vpa chronic treatment with vpa is commonly associated with weight gain, which potentially has important health implications, in particular increased central fat distribution. a positive correlation between chronic treatment with vpa and increased abdominal weight as well as increased blood pressure was recorded previously in human patients (20). an association was found between vpa induced weight gain and insulin resistance, hyperinsulinemia, hyperleptinemia and leptin resistance. furthermore, the patients who had vpa induced weight gain were more likely to develop metabolic syndrome and dyslipidemia with associated long term vascular complications such as hypertension and atherosclerosis. in addition, they stated that long term vpa therapy carries a risk for atherosclerosis because of an accumulation of oxidative stress in combination with elevations of uric acid and homocysteine. another experiment in human patients showed that vpa induced weight gain in the experimental group taking valproate but noted weight gain was not due to decreased physical activity. in these participants of the study, there was a noted increased motivation to eat and decreased glucose levels in the experimental group compared to control group (21). interestingly, quite opposite to these results in humans, khan at al. (22) in their recent work in animal models discuss that the reason for the weight increase with vpa therapy might be increased appetite and irregular thirst, and consequent over-consumption of energy-rich alimentary. also, they point out the dependence on the doses and duration of treatment, which changes the pharmacological signaling, differences in clinical and preclinical findings, as well as inter-species variability. the authors highlighted the role of histone deacetylases (hdacs) in insulin-resistance, gluconeogenesis and islet function, and formed a hypothesis that vpa treatment (in particular dose) might have a beneficial role in diabetic disorder. hdacs can modulate the expression of various genes, which directly or indirectly affect glucose metabolism. in their experimental work, khan et al. (22), proposed and recorded the anti-diabetic role of vpa by the modulation of insulin signaling and forkhead box protein o1 (foxo1)mediated gluconeogenesis in type-2 diabetic sprague-dawley rats. in the proposed model diabetes was induced by the combination of a high-fat diet and low dose streptozotocin, and vpa was given at the doses of 150 and 300 mg/kg/day. vpa treatment significantly reduced the plasma glucose, hba1c, insulinresistance, and the fat deposition in the brown adipose tissue, white adipose tissue and liver, which is comparable to metformin effects— which the authors used as a positive control to create equal effects. the treatment also inhibited the gluconeogenesis and glucagon expression and recuperated the histopathological changes in the pancreas and liver. detailed molecular mechanisms of the findings might elucidate the possible physiological mechanisms. seemedj 2017, vol 1, no. 1 the dual nature of the antiepileptic drug valproic acid… 80 southeastern european medical journal, vol 1, 2017. adipokines as expressed in the previous chapter, in recent years, research focused on adipokine signaling to elucidate the vpa effects in increased obesity. it seems that without doubt, vpa affects the neurohormonal regulation of appetite, as confirmed in both patients and animal models. weight gain during vpa treatment may be related to increases in leptin, insulin and neuropeptide y (npy) levels. tokgoz et al.(23), conducted a study on 20 prepubertal children which compared the bmi, leptin, insulin, npy, adiponectin and gherlin levels with lipid profiles, and caimt(carotid artery intima media thickness), before and after a treatment period with vpa of six to twelve months. the study aimed to evaluate and to determine whether these parameters indicate the development of early atherosclerosis. the treatment did not affect plasma ghrelin, adiponectin levels, lipid profiles, and caimt after 6 and 12 months of treatment compared with pretreatment values. although bmi and the appetite regulatory molecules were changed, early atherosclerotic changes were not triggered within a year of treatment (23). a similar study on vpa therapy, conducted earlier on 18 children (10.94 ± 3.78 years), showed, after 18 months of vpa exposure, an increase inthe serum leptin, insulin, and neuropeptide y (npy) levels, but also in the glucose, cortisol, galanin and ghrelin levels compared to the matching control. there was an increase of 2.3kg of body weight combined with an increase in named obesity signaling molecules in the treatment group compared to the control group (24).the findings of these two studies of decreased gherlin levels and increased weight gain are in stark contrast to the findings of the gungor et al. (25) group which found increased levels of gherlin in proportion to an increase in weight of prepubertal patients. meral et al. (26) included 44 children with idiopathic, generalized epilepsy treated with valproic acid (vpa) as an experimental group, and 40 children without therapy as the control group in their study (26). neither the vpa treated group nor the control group showed any significant difference in terms of ldl cholesterol, total cholesterol and age. however, the vpa treated group subjects had significantly higher bmi-sds as well as higher levels of visfatin, apelin, and triglycerides, but lower levels of adiponectin and hdl levels than the control subjects. the group concluded that visfatin, adiponectin and apelin can be considered as potential regulators of fat and glucose metabolism during valproic acid therapy (26).grosso et al.(27) found that patients with vpa associated obesity had high concentrations of leptin in their blood. they did not find any differences in leptin concentration between patients who had vpa induced obesity and obese controls during their experiment. li et al. (28) found that vpa can increase serum lipid levels in both juvenile and adult rats, but that higher levels of lipids could be found in juvenile rats (28). gherlin is affected by valproic acid treatment and there are potential effects of such interactions on weight gain.a study on 35 pediatric patients aged three to fifteen years were evaluated for gherlin, leptin, c-peptide, insulin, insulin like growth factor1, insulin like growth factor binding protein3 and glucose levels.serum gherlin levels were increased significantly with a negative correlation with insulin like growth factor-1 and insulin like growth binding protein 3 in the prepubertal group at six months of treatment. thus, the weight gain caused by valproic acid could be linked to the increased levels of gherlin levels in the early treatment period (25). polycystic ovarian syndrome (pcos) a higher occurrence of polycystic ovarian syndrome (pcos) compared with other antiepileptic drugs is one of the major adverse drug reactions reported in women treated with vpa (29;30). several molecular mechanisms could account for this epidemiological appearance. vpa treatment can be connected to hyperandrogenism, hyperandrogenemia, oligoovulaton, the appearance of pco on anultra-sonogram, elevated levels of testosterone, and irregular menstrual cycles. the incidence of occurrence of pcos in women taking vpa is 1.95 times higher than in women seemedj 2017, vol 1, no. 1 the dual nature of the antiepileptic drug valproic acid… 81 southeastern european medical journal, vol 1, 2017. being treated with other antiepileptics (31). it seems that vpa treatment during pregnancies possesses a significant risk of teratogenic effects based on a survey where a 15 out of 229 (6.6%) women prescribed valproate gave birth to a child with a major congenital malformation (29). since testosterone levels slightly increase, and progesterone and estradiol levels decrease over a longer treatment period with vpa, it is believed that such an imbalance induces polycystic ovarian changes and menstrual suspensions (amenorea) that later lead to polycystic ovarian syndrome (32). indeed, some authors, who also recorded increased levels of testosterone in women treated with vpa for bipolar disorder, proposed that a broad inhibitory action on glucuronidation systems and on cytochrome systems causes high concentrations of testosterone, dehydroepiandrosterone sulfate, and androstenedione. vpa treatment over a longer period of time is associated with increased levels of testosterone. it is also associated with a development of menstrual abnormalities. there is a significant correlation between vpa treatment and a reduction in the levels of mrna encoding estrogen receptor alpha (er alpha), which causes a lack of er alpha protein in breast and ovarian cell lines. beside the regulation of sex physiology and menstrual abnormalities, the weight gain and osteoporosis could be a result of the lack of estrogen signaling because of the clearance of er alpha protein in cell lines (33). furthermore, beside the vpa interference with the peripheral endocrine hormones, at the central regulatory level of the hypothalamushypophysis axis, vpa also causes decreased excretion of lh (luteinizing hormone), fsh (follicle stimulating hormone) and prolactin. together, the steroid sex hormone imbalance, the hyperinsulinemia and obesity in humans, which occur due to vpa treatment, further contribute to an increase in sexual dysfunctions (34). impairment of synthesis of steroid hormones mechanisms of ovarian toxicity include a possible disruption of the pathways for the synthesis of sex hormones as a major cause in the development of pcos. brion et al. (35) expressed that vpa may increase mitochondrial cholesterol transport by a mechanism independent of the steroidogenic acute regulatory protein, as proven in an experimental model. some authors explain the hormonal imbalance as a direct consequence of differential activity onthe expression of cyp enzymes involved in hormone synthesis, which impairs the conversion of testosterone to estradiol (36, 37). a suppression of aromatase expression takes place in granulosa cells as an answer to treatment with vpa. for example, the follicular development of 14-day-old rats was suppressed, and testosterone, estradiol, androstenedione, and the combined levels of all steroid hormones tended to decrease over time with exposure to vpa (38). gustavsen et al. (37), found in vitro, that the expression of genes coding for enzymes early in steroidogenesis was downregulated. such changes did not occur with the use of other antiepileptics (37). the suppression of aromatase expression means that the synthetic pathway from cholesterol to estradiol, including the de novo synthesis of cholesterol, is suppressed (38). contrary to these in vivo results in isolated mitochondria, in vitro, vpa stimulates exogenous cholesterol conversion to progesterone (38). this indicates that the complex physiological mechanisms do not fulfill the whole picture of physiological interactions as in vivo. the majority of the mechanistic studies on stereidogenesis of sex hormones and ovarian hyperandrogenism rely on in vivo and in vitro investigation of vpa exposure only, and measurements of cyp conversion in patients is scarce. there are inconsistencies in the literature regarding the effects of vpa on the female reproductive steroidogenesis, as pointed out by glister et al. (39) in their review of literature on the subject. in vivo in rodent models, glister et al. (39) mention both the increase in the number of seemedj 2017, vol 1, no. 1 the dual nature of the antiepileptic drug valproic acid… 82 southeastern european medical journal, vol 1, 2017. follicular “cysts” and total ovarian weight with decreased plasma testosterone level, then list experiments with no effect on serum androgen levels. however, reduced serum estrogen levels affect tthe androgen/estrogen ratio, where androgen hormones become relatively higher. interestingly, glister (39) mentions the experiment on primates where vpa treatment in a rhesus monkey with normal cycling had no effect on androgen levels or ovarian morphology. the authors (39) further list similar inconsistencies in the in vitro studies with some experiments: (i) recordings of increased ovarian androgen synthesis and an increased transcription of steroidogenic genes, (ii) an inhibitory effect of vpa on hcg-induced androgen secretion, (iii) increased basal and lhstimulated androgen secretion or decreased lh-stimulated androgen secretion and reduced basal and fsh-induced estradiol secretion. in their own experiment (39) in primary bovine theca (tc) and granulosa (gc) cells (a model closely relating human cycling), they exposed tc to vpa (7.8–500 µg/ml) with/without lh and gc with/without fsh or igf analogue. in theca cells, vpa reduced basal androstenedione secretion by 70% and in vpa/lh-induced theca cells by 93%. cyp17a1 mrna was reduced by more than 99% and lhr, star, cyp11a1 and hsd3b1 mrna was also lower. vpa only reduced theca cells progesterone secretion induced by the highest (luteinizing) lh dose. at higher concentrations (125–500 µg/ml) vpa inhibited basal, fshand igf-stimulated estradiol secretion in granulose cells without affecting progesterone secretion. vpa reversed fsh-induced upregulation of cyp19a1 and hsd17b1 mrna levels. vice versa, vpa inhibits both lh-dependent androgen production and fsh/igf-dependent estradiol production. the authors proposed that the named changes are consequences of the hdac inhibitory properties and conclude that the vpa has a direct stimulatory action on theca cell androgen production (39). alzheimer’s disease and vpa, new perspectives in recent work, evidence was gathered that besides gaba promoting effects, vpa treatment has antiapoptotic and protective effects in neural tissue (figure2), though as with steroidogenesis, mostly in model experiments. the deinhibition of histone acetylation caused by histone deacetylases (hdac s) inhibitors could contribute to the recovery of learning and memory in rats. histone hypoacetylaton of lysine residues contributes to cognitive impairments in alzheimer’s disease (ad).vpa can significantly elevate histone acetylation trough hdac activity inhibition. experiments showed that vpa treatment can boost the long-term recognition memory and spatial learning memory in ad transgenic mice. as such vpa could significantly improve cognitive function in ad (40, 41). experimentally, vpa ameliorates spatial memory impairment and amyloid beta deposition in transgenic mice (42). vpa treatment caused a decrease in senile plaque formation, and amyloid beta 40 and amyloid beta 42 accumulations. several studies, including those by longet al. (43) in 2016, proposed that gender differences play a role in determining how well vpa affects the treatment of alzheimer’s disease. it appears that gender differences play a role in the vpa effects on ad, since these effects were more notable in the male than in the female ad mice (43). as a histone deacetylase inhibitor, vpa is able to upregulate neprilysin (nep) expression and activity in human neuroblastoma sh-sy5y cell lines, which usually express very little nep protein. upregulation of expression and activity of nep in the rat hippocampus was, also, observed following i.p. injections of vap to rats. nep is an amyoloid degrading enzyme which in the healthy brain maintains amyloid beta levels at physiologically low concentrations. the activity and expression of these enzymes decreases with age and, with some other pathological conditions, predisposes to late onset of ad (40). the synapse damage caused by amyloid beta protein was reduced by pretreatment with physiologically relevant concentrations of vpa seemedj 2017, vol 1, no. 1 the dual nature of the antiepileptic drug valproic acid… 83 southeastern european medical journal, vol 1, 2017. (10 μm). vpa also decreased synaptic damage caused by other neurodegenerative associated proteins such as alpha synuclein, which is linked to lewy body dementia and parkinson’s disease (44). vpa treatment inhibited the activity of gsk3beta, decreasing hyper phosphorylated tau by lower phosphorylation, as seen in the transgenic mice (45). one of the main reasons of neuronal loss in ad is apoptosis of neurons. it seems that in neural tissue, vpa promotes the reduction of apoptosis by blocking and decreasing apoptotic signals and increasing survival (46). evidence for reduction of apoptotic signals includes a significant reduction in the expression of interleukin 1-beta (il-1ß) and the tumor necrosis factor alpha (tnf-α), as well as the micro and astrogliosis in the hippocampus and cortex of app/ps1 transgenic mice (43). besides the reduction of antiapoptotic signals, these results imply that vpa has anti-inflammatory properties in the brain that are opposite of the effects it has in the liver. further evidence of a decrease of apoptotic signaling in the mouse ad model showed that vpa acted via the suppression of both the mitochondrial and the endoplasmic reticulum pathways of apoptosis, by downregulating the expression of caspase 3, caspase 9, caspase 12, and by reducing the level of cytochrome c and bax. antiapoptotic protein bcl 2 expression was increased, intracellular levels of ca2+ decreased, and the mitochondrial membrane potential was elevated (46).these are yet other effects of vpa which seem to be completely opposite of those that are occurring in the liver where apoptosis inducing effects were demonstrated, as described previously in the text. most interestingly, beside the antiapoptotic effects, the proliferative effects in neurons, increase in synaptogenesis and novel connectivity between neurons are also important novel physiological roles of vpa in neural tissue. experimentally, vpa caused an increase of creb and bdnf expression, causing accelerated neurite outgrowth, modification of synaptic structure and improvement of behavioral deficits in ad mouse models. accumulating evidence supporting such research included reports where vpa was able to induce map 2 gene expression, which mediated the process of de novo re-arborisation and neurite outgrowth of neurons. these functions add to the process of successful neuronal re-wirings (47).vpa treatment includes the activation of regulatory pathways that enhance neurogenesis and suppress gliogenesis. genes which encode the transcription factors (tfs) that specify neuronal cell fate, including mef2d, myt1l, neurod1, pax6 and tbr1, and their target genes, are induced by vpa (48). neural stem cells (nscs) derived from niemann-pick type c disease (npc) mice, a neurodegenerative and lipid storage disorder for which no effective treatment is known, showed impaired selfrenewal and differentiation. vpa was able to induce neuronal differentiation and restore impaired astrocytes in nscs from npc1 (-/-) mice. increasing levels of cholesterol within nsc from npc1 (-/-) mice could be reduced by vpa. necessary neurotrophic genes (trkb, bdnf, mnsod, and neruod) were upregulated through the repression of the rest/nrsf and hdac complex by vpa treatment. these upregulated neurotrophic genes were able to enhance neural differentiation and cholesterol homeostasis in neural stem cells from npc1 (-/) mice (49). such protective and beneficial role in ad, tau and amyloid accumulation will be the future of vpa research. discussion and conclusion vpa is a short, branched chain fatty acid with strong anticonvulsant properties. it has diverse effects in different tissues, with opposing mechanisms of action that seem to be dose and exposure-time dependent (figure 1). the number of papers published so far (table 1), including approximately several hundred papers each year in the last decade, in all areas of physiological action, on both models and patients (table 2), prove that the research on vpa physiology still raises interest in basic physiological research. the literature cited in this work, divided by subject area of research and seemedj 2017, vol 1, no. 1 the dual nature of the antiepileptic drug valproic acid… 84 southeastern european medical journal, vol 1, 2017. type of research (models and patients) (table 2), is mainly from the last few years of research with only a few articles extended to the previous decade. from the comparison of cited physiological mechanisms, several major questions arise that will shape further research. although a relationship between vpa therapeutic mechanisms and gaba action is generally accepted, the mechanisms of its adverse reactions in other tissues have not been fully elucidated. the major questions that will impact future research directions are whether there is a unique fundamental physiological mechanism that triggers different reactions in the tissues with opposing action, or whether the different tissue responses are a consequence of diverse target pathways induced by vpa. answering this fundamental question will direct table 2. cited literature on valproic acid and/or valproate, divided into categories by subjects of proposed physiological mechanisms and scientific research methods in vitro, in vivo and epidemiological studies on patients.. physiological parameter in vitro (cell culture) in vivo (animal models) human patients (case, clinical and epidemiological studies) no. of cited papers (total) doses and therapy 1;2;3;4;5;6;10 7 biotransformation 7;9;11;14 38 7;8;11;16 9 liver adverse effects 9;12;13;14;15;17;18 18;19 8;17 11 gonads, hormones, sexual physiology adverse effects 29;33;35;36;37;38 38 30;31;32;34 11 stereidogenesis adverse effects (biotrasformation enzymes in sex hormones biochemistry) 35;36;37;38;39 5 overweight, bmi, adiposity 16;20;23;24;25;27 6 triglicerides, fatty acids cholesterol (biochemical mechanisms, adverse effects) 18;49 18;19;28 16;26 7 adipokines 28 16;20;21;23;24; 25;26 8 glucose and insulin, energy metabolism 22 29 16;21;23;24;25 7 oxidative stress 13;14;15 3 neural tissue beneficial effects, synaptogenesis, neural growth and beneficial effects in alzeheimer disease models 44;49 40;41;42;43;44; 45;46;47;48 11 no. of cited papers (total) 30 17 37 seemedj 2017, vol 1, no. 1 the dual nature of the antiepileptic drug valproic acid… 85 southeastern european medical journal, vol 1, 2017. and focus the research toward a mechanistic explanation of adverse effects and hypothetical novel properties, such as a model based beneficial mechanism in alzheimer’s disease. the beneficial effects in neural tissue is mainly gained hypothetically in the models (table 2) and has yet to be proven in human subjects. the models based on a novel proposed beneficial mechanism in neurons and especially in the protective role in the forming of alzheimer’s disease should also investigate the potential of vpa to prevent the transmigration of accumulated tau and amyloid in already formed plaques within the neural tissue. research of such therapeutic potential in patients with early signs of plaque formation would be more important than vpa preventive potential, since it is highly unlikely that the healthy patients could be subjected to therapeutic exposure to vpa as a measure of prevention, even if the beneficial effects in prevention of the first steps of accumulation, and hyperphosphorilation of these molecules are proven in humans. the problem of weight gain and energy physiology caused by the treatment with vpa still remains unsolved and limits all beneficiary actions described in neural tissues. although noted in patients and animal models, the adverse drug effects on adiposity and lipid accumulation has not been clearly explained. it is believed that the hepatocyte mitochondrion disturbance is a major cause of imbalance of lipid metabolism in the liver and one of the causes of weight gain observed in patients treated with vpa. however, even though some genetic predispositions (such as polg mutation) have been detected, it seems from the cited references that the effects could be more epigenetic in nature, and future research should focus on designing experiments connecting targeted genetic predisposition (mutations) and epigenetics. furthermore, the hepatocyte lipid imbalance should be further investigated physiologically from the point of major conversing enzymes along the biochemical pathways of triglyceride, fatty acid and cholesterol synthesis/biochemical redistribution and lipoprotein synthesis and redistribution (for example acetyl-coa carboxylases 1 and 2 (acc1 and acc2), fatty acid synthetase (fas), stearoyl-coa desaturase-1 or carnitine/palmitoyl-transferase 1 etc.). references in this direction of research are missing. besides the proposed hepatic adverse drug reactions that influence lipid metabolism and transport, recently, the research concentrated on the role of appetite regulation pathways involving adipokines and other metabolic regulating hormones. this subject is abundant in the literature with both evidence from the patients and animal models. the exact question that future experimental design has to focus on would be whether adipokine disturbance (increase) is directly unleashed by vpa action as a cause or whether it is a consequence of adipocyte (and gi) reaction to differential fatty acids. there is a probability that adipokine activation and appetite enhancements are merely a consequence of disturbed lipid physiology within the adipocytes or hepatocytes. besides lipid physiology, due to the abnormal effects on energy, fatty acid, and cholesterol metabolism, vpa treatment is a risk factor for the development of other metabolic diseases with its associated complications such as atherosclerosis, and metabolic syndrome, while in the cns it has many beneficial effects. research should also focus on the physiological pathways of cholesterol transport and metabolism not only in experimental models (table 2), but in the liver, reproductive organs (stereidogenesis) and in neural cells of patients as well. it seems that in the last decade the research field of oxidative stress has been abandoned or reduced in vivo (table 2). in addition to the biochemical pathways of lipid metabolism, the cellular redox status of biochemical balance might also be important in contributing to the differential activation of physiological pathways of lipid metabolism and epigenetic changes that may enhance pathophysiological changes. thus, future studies on vpa physiology should neither neglect this area of research. within all prospect studies, special attention should be given to the doses of exposure. seemedj 2017, vol 1, no. 1 the dual nature of the antiepileptic drug valproic acid… 86 southeastern european medical journal, vol 1, 2017. in conclusion, although prescribed as an effective anticonvulsant, most of the pathogenic pathways of vpa’s unwanted and beneficial molecular effects are not fully understood and further studies and experiments are warranted. disclosures competing interests: none to declare. acknowledgements and funding this work is a part of the project of the croatian academy of science and arts “cholesterol metabolism and oxidative stress”methcholox2015”, led by the corresponding author and the project grant no. ip-2014-099730alztauprotect -"tau protein hyperphosphorylation, aggregation and transsynaptic transfer in alzheimer's disease: cerebrospinal fluid analysis and assessment of potential neuroprotective compounds" of the croatian science foundation, where the corresponding author participates as a collaborator. references 1. european medicines agency. valproate 2017. http://www.ema.europa.eu/ema/index.jsp ?curl=pages/medicines/human/referrals/v alproate/human_referral_000187.jsp (date last accessed 18. 04. 2017) 2. zaccara g, giannasi g, oggioni r, rosati e, tramacere l, palumbo p. challenges in the treatment of convulsive status epilepticus. seizure 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10.1016/j.biochi.2016.02.014. 23. tokgoz h, aydin k, oran b, kiyici a.plasma leptin, neuropeptide y, ghrelin, and adiponectin levels and carotid artery intima media thickness in epileptic children treated with valproate. childs nerv syst 2012;28(7):1049-53. doi: 10.1007/s00381-0121788-7. 24. cansu a, serdaroglu a, camurdan o, hırfanoğlu t, cinaz p. serum insulin, cortisol, leptin, neuropeptide y, galanin and ghrelin levels in epileptic children receiving valproate. horm res paediatr 2011;76(1):6571. doi: 10.1159/000327367. 25. gungor s, yücel g, akinci a, tabel y, ozerol ih, yologlu s. the role of ghrelin in weight gain and growth in epileptic children using valproate. j child neurol 2007;22(12):1384-8. doi: 10.1177/0883073807307096. 26. meral c, cekmez f, vurucu s, tascılar e, pirgon o, canpolat fe, ipcioglu om, aydemir g, aydınoz s.new adipocytokines (vaspin, apelin, visfatin, adiponectin) levels in children treated with valproic acid. eur cytokine netw 2011;22(2):118-22. doi: 10.1684/ecn.2011.0284. seemedj 2017, vol 1, no. 1 the dual nature of the antiepileptic drug valproic acid… 88 southeastern european medical journal, vol 1, 2017. 27. grosso s, luisi s, mostardini r, matera m, barlocco eg, casarosa e, balestri p, petraglia f.circulating levels of allopregnanolone, a neuroactive steroid, and leptin during treatment with valproic acid in children with epilepsy. neuroendocrinology 2011;93(3):159-64. doi: 10.1159/000321664. 28. li j, li d, huang sp. [effects of valproate acid on blood lipid, serum leptin and cerebral cortex in juvenile and adult rats]. zhongguo dang dai er ke za zhi 2010;12(6):479-82. chinese. information from abstract: https://www.ncbi.nlm.nih.gov/pubmed/20 540861 29. petersen i, collings sl, mccrea rl, nazareth i, osborn dp, cowen pj, sammon cj. antiepileptic drugs prescribed in pregnancy and prevalence of major congenital malformations: comparative prevalence studies. clin epidemiol 2017;9:95-103. doi: 10.2147/clep.s118336. 30. viswanathan lg, satishchandra p, bhimani bc, reddy jy, rama murthy bs, subbakrishna dk, sinha s.polycystic ovary syndrome in patients on antiepileptic drugs. ann indian acad neurol 2016;19(3):339-43. doi: 10.4103/0972-2327.179973. 31. hu x, wang j, dong w, fang q, hu l, liu c.a meta-analysis of polycystic ovary syndrome in women taking valproate for epilepsy. epilepsy res 2011;97(1-2):73-82. doi: 10.1016/j.eplepsyres.2011.07.006. 32. herzog ag. disorders of reproduction in patients with epilepsy: antiepileptic drug related mechanisms. seizure 2008;17(2):1119. doi: 10.1016/j.seizure.2007.11.007. review. 33. reid g, métivier r, lin cy, denger s, ibberson d, ivacevic t, brand h, benes v, liu et, gannon f. multiple mechanisms induce transcriptional silencing of a subset of genes, including estrogen receptor alpha, in response to deacetylase inhibition by valproic acid and trichostatin a. oncogene 2005;24(31):4894-907. 34. verrotti a, mencaroni e, cofini m, castagnino m, leo a, russo e, belcastro v. valproic acid metabolism and its consequences on sexual functions. curr drug metab 2016;17(6): 57381. 35. brion l1, gorostizaga a, gómez nv, podestá ej, cornejo maciel f, paz c.valproic acid alters mitochondrial cholesterol transport in y1 adrenocortical cells. toxicol in vitro 2011;25(1):7-12. doi: 10.1016/j.tiv.2010.08.006. 36. taubøll e1, gregoraszczuk el, kołodziej a, kajta m, ropstad e. valproate inhibits the conversion of testosterone to estradiol and acts as an apoptotic agent in growing porcine ovarian follicular cells. epilepsia 2003;44(8):1014-21. 37. gustavsen mw, von krogh k, taubøll e, zimmer ke, dahl e, olsaker i, ropstad e, verhaegen s.differential effects of antiepileptic drugs on steroidogenesis in a human in vitro cell model. acta neurol scand suppl 2009;(189):14-21. doi: 10.1111/j.1600-0404.2009.01206.x. 38. inada h, chihara k, yamashita a, miyawaki i, fukuda c, tateishi y, kunimatsu t, kimura j, funabashi h, miyano t.evaluation of ovarian toxicity of sodium valproate (vpa) using cultured rat ovarian follicles. j toxicol sci 2012;37(3):587-94. 39. glister c, satchell l, michael ae, bicknell ab, knight pg.the anti-epileptic drug valproic acid (vpa) inhibits steroidogenesis in bovine theca and granulosa cells in vitro. plos one. 2012;7(11):e49553. doi: 10.1371/journal.pone.0049553. 40. nalivaeva nn, belyaev nd, lewis di, pickles ar, makova nz, bagrova di, dubrovskaya nm, plesneva sa, zhuravin ia, turner aj.effect of sodium valproate administration on brain neprilysin expression and memory in rats. j mol neurosci 2012;46(3):569-77. doi: 10.1007/s12031-011-9644-x. 41. yao zg, liang l, liu y, zhang l, zhu h, huang l, qin c. valproate improves memory deficits in an alzheimer's disease mouse model: investigation of possible mechanisms of action. cell mol neurobiol seemedj 2017, vol 1, no. 1 the dual nature of the antiepileptic drug valproic acid… 89 southeastern european medical journal, vol 1, 2017. 2014;34(6):805-12. doi: 10.1007/s10571-0130012-y. 42. xuan ag, pan xb, wei p, ji wd, zhang wj, liu jh, hong lp, chen wl, long dh.valproic acid alleviates memory deficits and attenuates amyloid-β deposition in transgenic mouse model of alzheimer's disease. mol neurobiol 2015;51(1):300-12. doi: 10.1007/s12035-014-8751-4. 43. long z, zeng q, wang k, sharma a, he g. gender difference in valproic acid-induced neuroprotective effects on app/ps1 double transgenic mice modeling alzheimer's disease. acta biochim biophys sin (shanghai) 2016;48(10): 930-8. 44. williams rs, bate c.an in vitro model for synaptic loss in neurodegenerative diseases suggests a neuroprotective role for valproic acid via inhibition of cpla2 dependent signalling. neuropharmacology 2016;101:566-75. doi: 10.1016/j.neuropharm.2015.06.013. 45. long zm, zhao l, jiang r, wang kj, luo sf, zheng m, li xf4, he gq. valproic acid modifies synaptic structure and accelerates neurite outgrowth via the glycogen synthase kinase-3β signaling pathway in an alzheimer’s disease model. cns neurosci ther 2015;21(11):887-97. doi: 10.1111/cns.12445. 46. long z, zheng m, zhao l, xie p, song c, chu y, song w, he g. valproic acid attenuates neuronal loss in the brain of app/ps1 double transgenic alzheimer's disease mice model. curr alzheimer res 2013;10(3):261-9. 47. abdanipour a, schluesener hj, tiraihi t, noori-zadeh a.systemic administration of valproic acid stimulates overexpression of microtubule-associated protein 2 in the spinal cord injury model to promote neurite outgrowth. neurol res 2015;37(3):223-8. doi: 10.1179/1743132814y.0000000438. 48. higgins ga, georgoff p, nikolian v, allynfeuer a, pauls b, higgins r, athey bd, alam he. network reconstruction reveals that valproic acid activates neurogenic transcriptional programs in adult brain following traumatic injury. pharm res 2017. doi: 10.1007/s11095-017-2130-6. 49. kim sj, lee bh, lee ys, kang ks. defective cholesterol traffic and neuronal differentiation in neural stem cells of niemann-pick type c disease improved by valproic acid, a histone deacetylase inhibitor. biochem biophys res commun 2007;360(3):593-9. seemedj 2020, vol 4, no. 1 stigmatization of patients with viral infections and mass psychogenic illness 1 southeastern european medical journal, 2020; 4(1) invited review stigmatization of patients with viral infections and mass psychogenic illness 1 andrijana šantić 1,2, krešimir šantić 2,3, ivan radoja 2,4 , ivana jelinčić 1,5, dunja degmečić* 1,2 1 department of psychiatry, university hospital centre osijek, croatia 2 faculty of medicine, josip juraj strossmayer university of osijek 3 department of pediatrics, university hospital centre osijek, croatia 4 department of urology, university hospital center osijek, osijek, croatia 5 faculty of dental medicine and health , josip juraj strossmayer university of osijek *corresponding author: dunja degmečić, ddegmecic@gmail.com received: feb 29, 2020; revised version accepted: apr 2, 2020; published: apr 27, 2020 keywords: stigma, viral infection, infected patient, discrimination, isolation abstract background: the stigmatization of viral patients is primarily a negative attitude and a common opinion about people suffering from various infectious diseases of the viral etiology and their consequences. the belief and the attitude that individuals are not socially acceptable because potentially spreading contagion for the outcome has negative discrimination in our society. often such persons are excommunicated, which extends through all the social layers and ages. methods: the pubmed, sciencedirect, and springerlink databases were used for the research. keywords stigma, viral infection, infected patient, discrimination, isolation were entered to identify papers dealing with a viral infection, and stigmatization. results: after screening available databases in the last five years according to the selected keywords, the pubmed database yielded nine articles, the sciencedirect identified initially 87 articles, springerlink identified 42 articles. viral infection and stigmatization are of interest to numerous scientists. conclusion: the availability of information should create empathy and ensure openness to diversity. following the available literature, we understand that the biggest problem today is the social exclusion of people due to their viral illness, but equally the self-isolation of those infected due to the fear of being rejected and the misunderstanding of people from the environment. (šantić a, šantić k, radoja i, jelinčić i, degmečić* d. stigmatization of patients with viral infections and mass psychogenic illness. seemedj 2020; 4(1); 1-13) seemedj 2020, vol 4, no. 1 stigmatization of patients with viral infections and mass psychogenic illness 2 southeastern european medical journal, 2020; 4(1) introduction by observing the etymological side, the stigmatization in its widest sense originates from the word stigma (greek stigmatos), which in ancient greece referred to a sign made on the body of morally bad and less valuable persons, created as a consequence of stamping or indentation. often this was related to uncivilized persons who speak different languages, barbarians, savages, but also members of the same people, slaves, criminals, traitors, and others from the lowest layers of society, which was then socially acceptable to mark. the main function of such a marking principle was the exclusion of selected persons from society and their social degradation. the aim of such degradation was not to direct the physical punishment of the individual but to punish the person by losing their freedom outside the bars, i.e., in the surrounding world. we notice that it is a much more complex concept, closely related to several prejudices, discrimination, and is very often accompanied by the primarily imposed and subsequently internalized by the selfstigmatization. the advancement of civilization has changed social layers, but stigmatization has always been one step ahead of time. although the physical marking of persons as a method of humiliation may not be used nowadays, stigmatization is still present in our society (1-7). one of the most famous sociologists in recent times who dealt with the notion of stigmatization, erving goffman, described the term as "an attribute that makes a man socially different from others in the social category and lowers him to the status of infected or reduced," and stigmatized person as "the individual who disqualifies from full social acceptance." besides, he proposed to divide the stigmatization into three categories: physical differences (visible deficiencies, physical injuries, mutilations, tattoos), recognizable character deficiencies (mental and mental state, addiction, alcoholism, socially unacceptable behavior) and tribal stigmatization (inborn characteristics such as race, nation, religion (6, 8, 9). there are two forms of stigma that share three common characteristics: stereotypes, prejudice, and discrimination, as outlined in table 1. table 1. comparing and contrasting the definitions of public stigma and self-stigma comparing and contrasting the definitions of public stigma and self-stigma public stigma stereotype a negative belief about a group (e.g., dangerousness, incompetence, character weakness) prejudice agreement with belief and/or negative emotional reaction (e.g., anger, fear) discrimination behavior response to prejudice (e.g., avoidance, withhold employment and housing opportunities, withhold help) self-stigma stereotype a negative belief about the self (e.g., character weakness, incompetence) prejudice agreement with belief, negative emotional reaction (e.g., low self-esteem, low self-efficacy) discrimination behavior response to prejudice (e.g., fails to pursue work and housing opportunities) the first is the public stigma, which implies the opinion of the majority of the population towards a marginalized group, while the second selfstigma implies a negative opinion of a member of such a marginalized group (10-13). during our past, many diseases have played a very significant role in their devastating influence on society, and consequently, their negative psychological impact has grown. examples are numerous. starting with the plague, probably the first described pandemic of all time, which in history has repeatedly decimated humankind, seemedj 2020, vol 4, no. 1 stigmatization of patients with viral infections and mass psychogenic illness 3 southeastern european medical journal, 2020; 4(1) and it is related to tens of millions of victims. its most notable outbreaks were 430 bc, 165, 250, 541, 1350, and 1665. in the 11th century, leprosy was first described as a separate disease and its whole dark side (14). the beginning of the 19th century marks cholera, and at the end of it, we can see the first significant pandemics of a viral disease, influenza (15). first, those russian from 1889., then spain from 1918., and in the end, those asian from 1957. year. history then records a couple more significant outbreaks of the causative agent in the later years of the 20th and early 21st centuries (16). the modern age marks the exponential development of science, medicine, and the discovery of antibiotic therapy, so the previously mentioned threats of bacterial etiology, such as plague and leprosy, have become a minor adversary. however, the man did not become immune to disease outbreaks, and since 1981 a new topic has emerged in the professional literature, as well as in daily informative discussions, hiv aids (17). another disease that causes the virus and is followed by a series of more common occurrences of other viral pandemics. fortunately, they are no longer counted in the millions but in the hundreds and thousands, but they are discussed daily (18). it is necessary to take into account the dose of globalism, the ease of availability of new information, but also, more generally, better information for the population. looking empirically and with great caution, it is not surprising that the human race created an aversion to diseases and their causes. as beings at the top of the food chain, they remained an important link which, from the beginning, represented our most important natural enemy. therefore, it is not surprising that infectious patients carried a great social stigma. perhaps as pity, but more likely out of fear for their own lives, often infected persons are excommunicated, which extends through all the social layers and ages (16-18). methods literature research took place in february 2020. databases used in the search were pubmed, sciencedirect and springerlink. keywords stigma, viral infection, infected patient, discrimination, isolation were entered to identify papers dealing with a viral infection, and stigmatization. in the last five years, the pubmed database yielded nine articles. another 75 articles were found after using the option "similar results" with the first paper, and 40 articles were found for the second paper. inclusion criteria all articles containing the terms stigma, discrimination, virus infection, social exclusion, cohort, prospective in the title or abstract were included in the review. exclusion criteria were if a paper included animal research, a person under 19 years. six articles regarding studies ultimately met the criteria. the final results obtained after applying filters are shown in table 2. table 2. pubmed search methodology and results filters paper 1 paper 2 from 2015 until 2020 11 28 similar results 78 93 english language 77 89 sex female and male 61 75 age: persons 19 – 80+ 58 74 review of abstracts and title of the articles based on including criteria 5 27 seemedj 2020, vol 4, no. 1 stigmatization of patients with viral infections and mass psychogenic illness 4 southeastern european medical journal, 2020; 4(1) the same keywords were used to search the sciencedirect database, and 87 articles were identified initially, which include articles published from 2015 to 2020. the number of articles was further reduced to 30 by using the option "type of article –research" and to 10 by using the option "publication title." they publish in the journals journal of the association of nurses in aids care, international journal of drug policy, social science & medicine, applied nursing research, hiv & aids review, journal of substance abuse treatment, journal of theoretical biology, nurse education today, journal of adolescence and sexual & reproductive healthcare. after reading the titles and abstracts, three articles were selected. the keywords mentioned above were used for searching the springerlink database, where 45 documents were identified, which include documents published from 2015 to 2020. after applying the filter article, the initial number was reduced to 42 and then to 36 after using the filter "medicine & public health." further restriction of results accomplishes using the options "public health and english," which led to 14 articles. titles and abstracts were reviewed, and two articles were selected. discussion the stigmatization of hiv patients aids is a disturbed condition of the human immune system, a disease caused by hiv infection. it affects populations worldwide, is not limited to specific subgroups or regions, and attacks without warning. it estimates that around 37.9 million people are currently infected worldwide, of which as many as 1.7 million children are under the age of 15. table 3. new hiv infections by region, 2017−2018; source: unaids/who estimates; who hiv update july 2019 (available on: https://www.who.int/hiv/data/en/; accessed on february 2020) new hiv infections by region, 2017−2018 who region number of new hiv infections 2017 number of new hiv infections 2018 new hiv infections all ages (per 1000 uninfected population) 2017 new hiv infections all ages (per 1000 uninfected population) 2018 africa 1 100 000 1 100 000 1.15 01.07.20 [830 000-1 500 000] [800 000-1 500 000] americas 160 000 160 000 0.16 0.16 [120 000-200 000] [120 000-200 000] south-east asia 170 000 170 000 0.09 0.09 [120 000-210 000] [110 000-200 000] europe 170 000 170 000 0.19 0.19 [150 000-180 000] [150 000-190 000] eastern mediterranean 39 000 41 000 0.06 0.07 [24 000-64 000] [26 000-68 000] western pacific 120 000 120 000 0.06 0.06 [110 000-130 000] [110 000-140 000] global 1 800 000 1 700 000 0.24 0.24 [1 400 000-2 300 000] [1 400 000-2 300 000] seemedj 2020, vol 4, no. 1 stigmatization of patients with viral infections and mass psychogenic illness 5 southeastern european medical journal, 2020; 4(1) there is no specific therapy, and due to its characteristic structure and inability to retain antigenicity in living attenuated or dead organisms, the vaccine has not been discovered to date. taking into account the above facts, as well as the devastating and deadly consequences it carries, the fear of hiv infection is reasonably justified. however, knowing the methods of spreading the disease and the relatively successful prevention mechanisms, the stigmatization of the sick is often exaggerated and non-meaningful. there should also be considered clear data on the constant decline in the number of sufferers, with a fall of as much as 38% globally from 2001 to 2003, accompanied by a significant reduction in the number of deaths caused by aids (19). in 2018, about 770,000 people died from the effects of aids, compared to 1.2 million in 2010, and half a million more in 2004 (table 3) (20). the stigmatization of hbv and hcv patients unlike human immunodeficiency viruses, hepatitis viruses constitute a heterogeneous group of microorganisms that specifically attack liver tissue, causing acute and chronic diseases. hepatitis a and e viruses appear sporadically in the world; we call them diseases of "dirty hands," and the result of infection with these agents is mostly an acute disease that usually spontaneously passes through a period of 1 to 4 weeks (21, 22). hepatitis d is characteristic in that it never occurs independently as a cause of disease, but always accompanies an earlier infection with hbv as a superinfection, i.e., it causes chronic co-infection of hdv and hbv (23). for our research, however, we will pay special attention to those types of viruses that cause a majority of chronic infections and which pose a special challenge for physicians, and the health system a certain socioeconomic problem. this category includes hepatitis b and hepatitis c viruses, which have a long incubation period, after which they cause mild or no symptoms, and once transmitted to chronic disease in some patients can cause cirrhosis or hepatocellular carcinoma (24). their occurrence is not geographically specific, they are present in all parts of the world, and they can affect members of all social layers. it currently estimates that 250-290 million people are infected with hbv worldwide, and approximately 887,000 people die annually from the infection (26.) specific therapy does not exist; treatment is symptomatic and aims to prevent the development of liver cirrhosis and to prevent the occurrence of hepatocellular carcinoma. however, the hbv vaccine exists, and in combination with improved awareness and good preventive measures, it estimates that by 2030, the global hbv threat should reduce to the lowest possible level, which is also the world health organization's plan (26). unfortunately, despite such a good prognosis, the stigmatization of hbv patients persists. by contrast, the number of people infected with hcv in the world estimates to be four times lower, around 71 million, and the death rate is only half the yearly rate than that of hbv, approximately 400,000. there is certainly the fact that in most cases, hcv is transmitted in the vast majority of cases only by blood, and for hbv, there is data of transfer through bodily fluids. besides, a specific vaccine against hcv is still not available, so it is more difficult to prevent the disease itself if direct exposure occurs (2730). in addition to the health complications affecting the ill, an additional burden creates by stigmatization and discrimination (31, 32). the basis of stigmatization and, in this case, carries a high level of anxiety and excessive fear of transmission of infection, which consequently leads to social and economic discrimination and financial burdens for the individual and the economy as a whole. the basis of the prevalence of discrimination against these viral infections would also be education and accurately inform the entire world population (33). the stigmatization of ebola patients ebola virus disease is a rare disease of ebola virus infection. the risk of ebola virus infection is extremely small unless you have been in direct contact with the bodily fluids of people suffering seemedj 2020, vol 4, no. 1 stigmatization of patients with viral infections and mass psychogenic illness 6 southeastern european medical journal, 2020; 4(1) from ebola, i.e., living or dead animals (34,35). uncertainty among populations is given by the fact that it is possible to transfer infection by contact with bodily fluids or unprotected sex with patients who have completely recovered from the ebola virus disease. in particular, special warnings issues to persons traveling to countries where there is a risk of infection with ebola. the stigmatization of ebola patients is mentioned in several scientific articles (36-38). talking about the discrimination and stigmatization of ebola-infected in posts about survivors, up to 64% of articles in 2015 were explicitly stigmatized related to survivors (39). trough many articles, the main cause of any form of stigmatization and/or discrimination has been called fear of illness (40). what cited as the basic problem for survivors is dealing with the consequences, social and economic outcomes (loss of friends, loss of workplace), and experiencing psychological stress. often, these issues compound by the unreliability of the health system, inconclusive knowledge about treatments, and ways of transmitting infection with a lack of feedback and the effectiveness of destigmatizing measures (41, 42). the current epidemic mostly affects the democratic republic of congo with liberia, guinea, and sierra leone, with a survival rate of 53% (table 4) table 4. chronology of previous ebola virus disease outbreaks. latest numbers as of february 23, 2020. source: ministry of health, democratic republic of the congo (available on: https://www.who.int/emergencies/diseases/ebola/drc-2019; accessed in february 2020) chronology of previous ebola virus disease outbreaks. latest numbers as of february 23, 2020. year country evd cases deaths case fatality 2018-2020 the democratic republic of the congo zaire 3444 – ongoing 2264 65,74% 2018 the democratic republic of the congo zaire 54 33 61,00% 2017 democratic republic of the congo zaire 8 4 50,00% 2015 italy zaire 1 0 0,00% 2014 spain zaire 1 0 0,00% 2014 uk zaire 1 0 0,00% 2014 usa zaire 4 1 25,00% 2014 senegal zaire 1 0 0,00% 2014 mali zaire 8 6 75,00% 2014 nigeria zaire 20 8 40,00% 2014-2016 sierra leone zaire 14124* 3956* 28,00% 2014-2016 liberia zaire 10675* 4809* 45,00% 2014-2016 guinea zaire 3811* 2543* 67,00% seemedj 2020, vol 4, no. 1 stigmatization of patients with viral infections and mass psychogenic illness 7 southeastern european medical journal, 2020; 4(1) (43, 44). persons who are treated as survivors, cured of ebola, experience social isolation by people from their environment, and experience various forms of violence that, apart from psychic, also involve the extent of physical violence (45, 46). . the similarity of stigmatizing attitudes towards people suffering from hiv and ebola infection are numerous, from discriminatory racial attitudes, sexual orientation with irrational fears (47, 48). the fundamental difference in the stigmatization of the infections mentioned above is that the mortality of the ebola virus infection is higher compared to the affected population, it occurs within a shorter period, and therefore, the level of anxiety is even higher (49). the stigmatization of patients with respiratory infections two-thirds of all infections are inflammatory diseases of the respiratory system. the most common causes of respiratory infections are viruses, accounting for 85% of all acute respiratory inflammation (50). due to the lack of availability of effective antiviral drugs, treatment generally comes down to symptomatic. the basic mechanism of transmission is by dropthrough, more frequently occurring in the colder months with symptoms such as fever, cough, shortness of breath, muscle pain, fatigue, and general weakness (51). although most often, it is a common cold caused by rhinovirussians, more emphasis places on the flu virus due to the severe clinical images and possible mortality. complications of influenza in older age, cause the so-called. "excess mortality." for this reason, influenza in older age should be recognized as a serious disease, especially for those who already have impaired health. influenza viruses are constantly changing, with new strains appearing regularly. if you've had influenza previously, your body has already made antibodies to fight that particular strain of the virus. if future influenza viruses are similar to those you've encountered before, either by having the disease or by getting vaccinated, those antibodies may prevent infection or lessen its severity. but antibodies against flu viruses you've encountered in the past can't protect you from new influenza strains that can be very different immunologically from what you had before. there are three types of flu viruses: a, b, and c. type a and b cause the annual influenza epidemics that have up to 20% of the population sniffling, aching, coughing, and running high fevers. type c also causes flu; however, type c flu symptoms are much less severe (52, 53). the flu is linked to between 3,000 and 49,000 deaths and 200,000 hospitalizations each year in the united states. the seasonal flu vaccine was created to try to avert these epidemics. type a flu virus is constantly changing and is generally responsible for the large flu epidemics. type b flu may cause a less severe reaction than type a flu virus, but occasionally, type b flu can still be extremely harmful. influenza type b viruses are not classified by subtype and do not cause pandemics. type c flu viruses do not cause epidemics. all flu vaccines protect against three influenza viruses: one influenza a (h3n2) virus, one influenza a (h1n1) virus, and one influenza b virus. the avian influenza virus causes bird flu. birds can be infected by influenza a viruses and all of its subtypes. birds are not capable of carrying either type b or c influenza viruses (54, 55). the flu season in the republic of croatia is expected to start at the end of november and ends in late march with roughly an affected population of 75 000 people (figure 1 and 2). since it is a serious illness, vaccination is highly recommended. seemedj 2020, vol 4, no. 1 stigmatization of patients with viral infections and mass psychogenic illness 8 southeastern european medical journal, 2020; 4(1) figure 1. the incidence rate of influenza affected patients by age groups in the republic of croatia on 100 000 inhabitants in season 2019./2020. available on: croatian institute of public health, https://www.hzjz.hr/; accessed in february 2020 figure 2. influenza prevalence per week in the republic of croatia in the season 2017/2018, 2018/2019, and 2019/2020. available on: croatian institute of public health, https://www.hzjz.hr/; accessed in february 2020 speaking of respiratory viral infections, we must not forget a microorganism that has occupied the public of the whole world and has become a media and infectious phenomenon in 2019. and in 2020. year, the covid-19 – coronavirus disease 2019. otherwise, it belongs to a group of viruses spread among humans as well as other mammals and birds. after infection, it can affect the respiratory, gastrointestinal, liver, and neurological systems, with consistent symptomatology. six known types of the virus affect humans, and in general, all manifest by seemedj 2020, vol 4, no. 1 stigmatization of patients with viral infections and mass psychogenic illness 9 southeastern european medical journal, 2020; 4(1) general infectious symptoms resembling influenza above, and without precision diagnostic technique, it is not possible to distinguish them by the clinical picture. the covid-19 causes milder respiratory symptoms in most cases. however, there are clear indications of complications and reported deaths (figure 3) (56). figure 3. epidemic curve of covid-19 cases (n=338) identified outside of china by date of onset of symptoms and likely exposure location, february 27, 2020. available on: https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200227-sitrep-38covid-19.pdf?sfvrsn=9f98940c_2; accessed in february 2020 at this time, while the pandemic is still ongoing, the total number of patients is growing worldwide, with a more and more deaths as a result of complications. at the time of writing this paper number of cases in croatia is growing (18). mass psychogenic illness the significant financial and psychological problem of today's time represents mass psychogenic illness. although attention often does not focus on this issue, it leaves significant consequences for the management of several systems starting from the health system, continuing to the educational and internal control of the functioning of states. it defines as the rapid spread of clinical signs of illness and symptoms among members of certain groups that manifests through disorders of mental state, physical functioning, and the presentation of numerous somatoform complaints that do not have a real organic basis (57). the trigger is sufficient (bad smell, bad taste, unknown sound, suspicious substance) that will convince the individual or the group that it has been exposed to a particular hazard and to experience selfsuggestive symptoms of the disease. specifically, the index case may have an organic problem, but later cases do not necessarily have to be affected by the cause. it is interesting that it affects women more often and manifests in symptoms of "phantom disease" with the onset of anxiety, social isolation, nausea, headache, abdominal pain, fainting, chest pain, weakness, and hyperventilation. following the available literature, the phenomenon of mass hysteria is most often associated with persons exposed to different stressors, certain structural seemedj 2020, vol 4, no. 1 stigmatization of patients with viral infections and mass psychogenic illness 10 southeastern european medical journal, 2020; 4(1) characteristics of personality, infrequently socioeconomic status, and individuals who have historically experienced trauma or abuse. the contribution to the development of this phenomenon is media reports with inconsistent and incomplete information filled with sensational concepts, thus losing the primary objective of informing and educating the public about medical information that may consequently harm news consumers by misleading and misinforming (58). as an example of the mass psychogenic illness occurring in a girls' high school at gopalganj in bangladesh in april 2013, which resolves with a rapid coordinated response within the affected population (59). an example of this form of mass hysteria can also mirror infections caused by viruses. fear of the unknown, insufficient information of the population, and the unwillingness of the entire social system can lead to the development of this phenomenon with the subsequent development of stigmatizing attitudes towards the seriously affected (18, 60). conclusion any stigmatization entails discrimination, especially if the original cause is long-lasting or incurable, which in turn harms the emotional state of the individual, which ultimately leads to self-stigmatization, deepening feelings of rejection, and aggravating the previously disturbed condition of the individual. in modern society, the attitude towards the diseased characterizing incomparably greater humanity than it was during the history. the quality of treatment, administration more effective, and every day more advanced pharmacotherapy, as well as the more efficient healthcare system, have led to increasing awareness and social acceptance of the patient. the level of awareness of certain diseases is increasing day by day, partly due to excellent information, but also high-quality health education of the population. patients are no longer treated as the less valuable beings, nor are they physically marked or punished. however, certain stigma in individual clinical branches persists. perhaps as a consequence of fear or compassion for the sick, it will hardly be eradicated as such for much longer. one stamp from the past may diminish over time, but unfortunately, the future always brings some new ones. collective psychogenic illness has been reported in literature since medival times. everyone has deep personal experience of panic. epidemic hysteria is a fascinating phenomenon, one that has occured for centuries and is likely to continue to occur. during times of threat, anxious public needs to feel reassured and protected and people look to authority figures to take control and provide that reassurance. public health agencies with planned, well coordinated, strategic approach will help reduce societal vulnerability to mass hysteria and limit the „contagiousness“ of such an event. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. smith ra, hughes d. infectious disease stigmas: maladaptive in modern society. commun stud. 2014; 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[published correction appears in plos med. 2005; 2(8):e321]. plos med. 2005; 2(7):e215. doi:10.1371/journal.pmed.0020215 59. tarafder bk, khan ma, islam mt, mahmud sa, sarker mh, faruq i, miah mt, arafat sm. mass psychogenic illness: demography and symptom profile of an episode. psychiatry j. 2016; 2016:2810143. doi:10.1155/2016/2810143 60. de wilde ah, snijder ej, kikkert m, van hemert mj. host factors in coronavirus replication. curr top microbiol immunol. 2018; 419:1–42. seemedj 2021, vol 5, no. 2 students during the covid-19 pandemic 38 southeastern european medical journal, 2021; 5(2) short communication attitudes and behavior of biomedical students in comparison with other students during the covid-19 pandemic 1 matea smajić *1, petra smajić 2, lada zibar 1,3 1 faculty of medicine, josip juraj strossmayer university of osijek, osijek, croatia 2 clinical hospital centre osijek, department of infectious diseases, osijek, croatia 3 merkur university hospital, department of nephrology, zagreb, croatia *corresponding author: matea smajić, matea.smajic@gmail.com received: aug 25, 2021; revised version accepted: nov 4, 2021; published: nov 26, 2021 keywords: medical students; covid-19; physical activity; vaccination abstract aim: since its beginning, the covid-19 pandemic has affected many people’s usual activities and lifestyle, including croatian students’ lives. the aim of the study was to examine whether the attitudes and behaviour of students in the biomedical (b) field differed from those in other (o) fields at the josip juraj strossmayer university of osijek (jjsuo). the hypothesis was that b students would behave more responsibly. materials and methods: a 10-question anonymous online survey on attitudes and behaviour related to the covid-19 pandemic was designed and conducted. the research was carried out in november 2020 and it included a total of 348 students (46 % of b students) at the jjsuo. the data were statistically processed by the ibm ® spss ® statistics 25.0 software at the statistical significance level of p < 0.05. results: twenty-five percent of b students and 11.17 % of o students (p = 0.001) responded that they did not go to nightclubs, in accordance with the civil protection headquarters’ recommendations. regarding their indoor socialising, 24.38 % of b students and 45.21 % of o students behaved the same as before the pandemic (p < 0.01). a total of 63.13 % of b and 39.36 % of o students (p < 0.01) responded that they would receive a vaccine against sars-cov-2. conclusion: b students behaved more responsibly than o students by reducing their socialising and going to crowded places, probably because of their education and awareness of the severity of covid-19. accordingly, more of them were willing to get the vaccine against sars-cov-2.. (smajić m, smajić p, zibar l. attitudes and behavior of biomedical students in comparison with other students during the covid-19 pandemic. seemedj 2021; 5(2); 38-43) seemedj 2021, vol 5, no. 2 students during the covid-19 pandemic 39 southeastern european medical journal, 2021; 5(2) introduction since the world health organization declared covid-19 a pandemic in march 2020 (1), people’s usual activities and lifestyle have fully transformed. since social gatherings have been restricted ever since then (2), the pandemic has exerted a huge impact on everyone’s day-today life, especially on young people’s social life. in the summer of 2020, croatia did not have high numbers of daily covid-19 cases (3). however, by the beginning of october, the numbers started to rise once again (3). due to alleviated restrictions in that transitory period (4), people in croatia prevented the coronavirus transmission primarily by their own behavior. therefore, we decided to examine the attitudes and behaviour of students at the josip juraj strossmayer university of osijek during the ongoing pandemic. the students were divided into two groups, biomedical students (b) and other (o) students, and the hypothesis was that b students would behave more responsibly. materials and methods in november 2020, a 10-question anonymous online survey on attitudes and behaviour related to the covid-19 pandemic was designed and conducted using google forms. it included a total of 348 students at the josip juraj strossmayer university of osijek, with 45.98 % of b students and 54.02 % of o students. of the total number of respondents, 70.11 % were female and 29.89 % were male. respondents were aged 18 or above, opted in to the study voluntarily and were required to give their informed consent before starting the survey. their habits during the pandemic were examined, including going to nightclubs, socialising indoors and going to a gym. we also considered whether they or their families had tested positive for covid-19 on a polymerase chain reaction (pcr) test. in addition, we asked for their general opinion about the vaccine and checked if they would get a vaccine once available on the market. statistical anaylsis chi-square test was used to examine the difference between the two groups. the data were statistically processed by the ibm®spss® statistics 25.0 software. statistical significance level was set to p < 0.05. results twenty-five percent of b students and 11.17 % of o students (p = 0.001) responded that they did not go to nightclubs, in accordance with the civil protection headquarters’ (cph) recommendations. a total of 65.8 % of both b and o students acknowledged that they went out less and more carefully (table 1). there was no statistically significant correlation between going out and considering whether they or their families had tested positive for covid-19. regarding their indoor socialising, 24.38 % of b students and 45.21 % of o students behaved the same as before the pandemic (p < 0.01). only 8.05 % of all students claimed that they did not socialise indoors at all, in accordance with the cph’s recommendations (table 1). once again, there was no statistically significant correlation between the students’ or their families’ positive tests for covid-19 and the students’ decisions about socialising indoors. a total of 35.34 % of b students and 54.74 % of o students stated that the level of their sports and recreational activities was the same as before the pandemic, while 64.66 % of b students and 45.26 % of o students answered that they performed recreational activities in accordance with the cph’s instructions and measures (p < 0.01). for the purpose of this comparison, 95 students who did not exercise were excluded (table 1). . seemedj 2021, vol 5, no. 2 students during the covid-19 pandemic 40 southeastern european medical journal, 2021; 5(2) table 1. going to nightclubs, socialising indoors and doing sports activities during the covid-19 pandemic (n = 348) table 1. question o students b students n % n % p* going to nightclubs i do not go to nightclubs in accordance with the cph’s recommendations. 21 11.2 40 25 0.001 i go out less and more carefully. 128 68.1 101 63.1 i go out the same as before the pandemic. 39 20.7 19 11.9 indoor socialising i do not socialise indoors in accordance with the cph’s recommendations. 12 6.4 16 10 i socialise indoors less and more carefully. 91 48.4 105 65.6 < 0.01 i socialise indoors the same as before the pandemic. 85 45.2 39 24.4 sports activities i do sports activities in accordance with the cph’s instructions and measures. i do sports activities the same as before the pandemic. 62 75 45.3 54.7 75 41 64.7 35.3 < 0.01 cph – civil protection headquarters, b – biomedical students, o – other students seemedj 2021, vol 5, no. 2 students during the covid-19 pandemic 41 southeastern european medical journal, 2021; 5(2) a total of 63.13 % of b and 39.36 % of o students (p < 0.01) responded that they would get a vaccine against sars-cov-2 once it was available. general opinions about vaccination given by students who would not get the vaccine were mostly related to their doubts about the effectiveness of the vaccine and their thoughts on vaccinating only high-risk groups. discussion as expected, there was a statistically significant difference between biomedical students and students from other fields in terms of following the cph’s measures. even though there was a significantly higher proportion of b students who did not go to nightclubs in comparison with o students, it was still expected that the percentage of b students who would not go to nightclubs in accordance with the cph’s recommendations would be much higher than 25 %. it must be taken into account that the research was conducted between late october and early november 2020, when croatia (population of 4 million) had over 15.000 covid19 cases per week (5). nonetheless, cafés, restaurants and nightclubs were still open, although their hours of operation were limited (6). on 12 november, there were 3,082 covid-19 cases recorded in croatia, which was the largest number of daily cases up to that moment (7). on 20 december, exactly a month after all the cafés, restaurants and nightclubs had been closed, 1,975 new daily cases were confirmed (8). following a slight drop in the number of daily covid-19 cases after their closure, it was concluded that nightclubs were one of the riskiest places for the coronavirus transmission. at the time of the research, only recommendations and moderate measures were imposed on indoor socialising (9). considering that, it is not surprising that a total of 124 students (34.64 %) did not follow those recommendations. however, when it comes to this issue, b students were still much more responsible than o students. the results showed that three-quarters of b students were aware of the fact that the coronavirus spreads easily in enclosed spaces, as well as of the risk they would pose to mutual families visiting and gathering indoors with their own family. the vast majority of b students exhibited responsible behavior as regards sports and recreational activities as well. more than half of b students claimed they performed recreational activities in accordance with the cph’s instructions and measures, meaning they worked out either at home or outdoors. the proportion of b (72.5 %) and o (72.8 %) students who were physically active (whether at a gym or at home/outdoors) at the time of the research was almost equal. these results differed from the results of a research by j. steffen et al., who reported that medical students were much more physically active during the pandemic compared to non-medical students (10). accordingly, the students in this study did not reduce their sports activities during the pandemic, unlike students of several other studies. in a survey by g. a. zello et al., 90 % of the students reduced their physical activities during the pandemic (11). moreover, in an international study by ammar et al., it was noted that the frequency, duration and intensity of physical activities decreased by 35 %, 34 %, and 42.7 %, respectively (12). the differences noticed between this and other studies can be explained by the fact that gyms and fitness centres were not shut down at the time of this research, unlike in the two aforementioned studies. as expected, a significantly larger number of b students expressed a positive opinion about being vaccinated against sars-cov-2. these results were mostly related to their education and knowledge about vaccine mechanism and effectiveness, which was confirmed by their opinions about the vaccine at the end of the survey. on the other hand, the most frequent reasons that o students pointed out as the reasons not to get vaccinated related to their fear of the short amount of time to develop the vaccine and their opinion about vaccinating only high-risk groups. seemedj 2021, vol 5, no. 2 students during the covid-19 pandemic 42 southeastern european medical journal, 2021; 5(2) conclusion this is the first-ever study examining the differences between biomedical students and students from other fields based on their usual activities during the pandemic. in addition, other studies have not yet examined students’ impression about the upcoming vaccine against sars-cov-2. as it was hypothesised, biomedical students adhered to the cph’s recommendations more and they were willing to get a vaccine against sars-cov-2 in a much higher proportion than students from other fields. the results could be associated with their long-term education in the field, along with their awareness of how easily the virus spreads among people. acknowledgement. this study was presented as an abstract and oral presentation at the international students’ conference oscon, faculty of medicine osijek, croatia 20.3.2021.. disclosure funding. no specific funding was received for this study. competing interests. none to declare references 1. world health organization. who directorgeneral’s opening remarks at the media briefing on covid-19 – 11 march https://www.who.int/directorgeneral/speeches/detail/who-director-general-sopening-remarks-at-the-media-briefing-on-covid19---11-march-2020 (2.8.2021) 2. centers for disease control and prevention – guidance for organizing large events and gathering https://www.cdc.gov/coronavirus/2019ncov/community/large-events/considerations-forevents-gatherings.html (2.8.2021) 3. koronavirus.hr – statistički pokazatelji za hrvatsku i eu https://www.koronavirus.hr/koronavirus-statistickipokazatelji-za-hrvatsku-i-eu/901 (2.8.2021) 4. vlada republike hrvatske – novi set mjera https://vlada.gov.hr/vijesti/bozinovic-novi-setmjera-donijet-ce-odredjena-postupnapopustanja/32173 (2.8.2021) 5. koronavirus.hr – covid–19 tjedno izvješće – 2. studenog https://www.koronavirus.hr/covid-19tjedno-izvjesce-2-studenog/851 (16.7.2021) 6. narodne novine – stožer civilne zaštite rh https://narodnenovine.nn.hr/clanci/sluzbeni/2020_11_128_2450.ht ml (2.8.2021) 7. ius-info – odluke stožera civilne zaštite rh u 2020. godini i relevantni propisi u uvjetima epidemija koronavirusa https://www.iusinfo.hr/aktualno/usredistu/41376#studeni2020 (19.7.2021) 8. ius-info – odluke stožera civilne zaštite rh u 2020. godini i relevantni propisi u uvjetima epidemija koronavirusa https://www.iusinfo.hr/aktualno/usredistu/41376#prosinac2020 (19.7.2021) 9. narodne novine – stožer civilne zaštite https://narodnenovine.nn.hr/clanci/sluzbeni/2021_06_67_1292.html (2.8.2021) 10. steffen j, schlichtiger j, brunner,s. huber bc. health promoting behaviour of medical versus nonmedical students during covid-19 pandemic: results from the cola cross-sectional study. j transl med 2021; 19:242. https://doi.org/10.1186/s12967-02102899-y 11. bertrand l, shaw ka, ko j, deprez d, chilibeck pd, zello ga. the impact of the coronavirus disease 2019 (covid-19) pandemic on university students’ dietary intake, physical activity, and sedentary behavior. appl physiol nutr metab. 2020; 46(3):265-272. https://doi.org/10.1139/apnm-20200990 12. ammar a, brach m, trabelsi k, chtourou h, boukhris o, masmoudi l, bouaziz b, bentlage e, how d, ahmed m, müller p, müller n, aloui a, hammouda o, paineiras-domingos ll, braakman-jansen a, wrede c, bastoni s, pernambuco cs, mataruna l, taheri m, irandoust k, khacharem a, bragazzi nl, chamari k, glenn jm, bott nt, gargouri f, chaari l, batatia h, ali gm, abdelkarim o, jarraya m, abed ke, souissi n, van gemert-pijnen l, riemann bl, riemann l, moalla w, gómez-raja j, epstein m, sanderman r, schulz sv, jerg a, al-horani r, mansi t, jmail m, barbosa f, ferreira-santos f, šimunič b, pišot r, gaggioli a, bailey sj, steinacker jm, driss t, hoekelmann a. effects of covid-19 home https://doi.org/10.1139/apnm-2020-0990 https://doi.org/10.1139/apnm-2020-0990 seemedj 2021, vol 5, no. 2 students during the covid-19 pandemic 43 southeastern european medical journal, 2021; 5(2) confinement on eating behaviour and physical activity: results of the eclb-covid19 international online survy. nutrients. 2020; 12(6):1583. doi: 10.3390/nu12061583.. 1 author contribution. acquisition of data: smajić m, smajić p, zibar l administrative, technical or logistic support: smajić m, smajić p, zibar l analysis and interpretation of data: smajić m, smajić p, zibar l conception and design: smajić m, smajić p, zibar l critical revision of the article for important intellectual content: smajić m, smajić p, zibar l drafting of the article: smajić m, smajić p, zibar l final approval of the article: smajić m, smajić p, zibar l statistical expertise (statistical analysis of data): smajić m, smajić p, zibar l seemedj 2021, vol 5, no. 1 medical vs surgical abortion 183 southeastern european medical journal, 2021; 5(1) review article medical vs surgical abortion. overview of european legislation and health care practice 1 francesca negro 1 *; maria cristina varone 1 *; antonella cotoia 2; renata beck 2 1 . department of anatomical, histological, forensic and orthopedic sciences, sapienza university of rome, rome, italy 2 anesthesia and intensive care unit, department of medical and surgical sciences, university of foggia, policlinico "auo riuniti", foggia , italy * equal contribution corresponding author: renata beck, beckrenata64@gmail.com received: oct 9, 2020; revised version accepted: feb 1, 2021; published: apr 28, 2021 keywords: abortion, legislation, mifepristone abstract introduction: abortifacient drugs, such as ru-486 or mifepristone, used in combination with a prostaglandin analogue (misoprostol) for the purpose of achieving medical abortion, have given rise to major legal, ethical and moral quandaries, which legislators all over europe have striven to overcome by reconciling the reproductive rights of women with those of dissenting medical personnel. materials and methods: we have conducted a comparison between international legislative approaches from the 1970s to 2020 upon the subject of voluntary abortion, with an eye on their applicability as well as other ethical concerns, supported by the analysis of the scientific debate on medical vs surgical abortion. results: the unresolved rift between the reproductive will of women and medical professionals’ claim to conscientious refusal to treat, i.e., refusal to perform abortions or to prescribe abortifacient medicine, in such overwhelming numbers in italy and elsewhere, has given rise to the impossibility of many women to terminate their pregnancies as they choose to. as a matter of fact, in 2018, only 64.9% of italian public hospitals were able to guarantee access to abortion services. hence, 35% of italian facilities fail to meet the standards set by law 194/78. conclusion: the authors have aimed to shed light on how medical abortion is to be preferred over a surgical one, and how major european countries have dealt with such an extremely thorny issue that has polarised the public opinion and scientific community members alike. (negro f; varone mc, cotoia a; beck r. medical vs surgical abortion. european overview of legislation and health care practice. seemedj 2021; 5(1); 183-190) seemedj 2021, vol 5, no. 1 medical vs surgical abortion 184 southeastern european medical journal, 2021; 5(1) introduction ru-486 (mifepristone) is an active antiprogesterone and antiglucocorticosteroid agent, generally used in combination with a prostaglandin analogue (misoprostol) in order to bring about a medical abortion during pregnancy. except for poland, ireland, and malta, where abortion is banned, access to the medication is regulated throughout the european union, albeit through varying, rather than uniform protocols, as reflected in table 1 (1, 2). much like in the eu, in the united states and several eastern european countries, as well as in india, china, and all countries where abortion is legal, mifepristone combined with misoprostol is the most widespread means to induce an abortion. the world health organization itself has deemed the drugs safe and effective (3, 4). table 1. comparison between voluntary termination of pregnancy vs medical abortion in diferent european countries country voluntary termination of pregnancy medical abortion austria legal within the first 3 months of pregnancy (20). legal and accessible. the regulation for mifepristone allows the drug to be administered only in medical facilities; medical abortion can therefore only be performed in hospitals (21). belgium legal within the twelfth week of pregnancy. available in belgium and given until up to 49 days of amenorrhea (22). bulgaria legal within the twelfth week of pregnancy (23). abortifacient drugs are not registered, thus illegal. croatia legal within the tenth week of pregnancy (24). medical abortion has been available since 2015. only recently has the croatian agency for medicinal products and medical devices (halmed) approved the drug combination used in medical abortion. yet, their administration is only allowed in hospitals accredited to perform abortions, with professional supervision. cyprus legal within the tenth week of pregnancy since 2018 (25). misoprostol is legally usable for termination of pregnancy. czech republic legal within the first 3 months of pregnancy (26). legal upon demand since 2013. denmark legal within the twelfth week of pregnancy since 1973 (27). medical abortion is used until end of 8th week. surgical abortion may be chosen until end of 12th week. until 8th week, most abortions are medical. overall, approx. 40% of all the abortions are medical and approx. 60% of all the abortions are surgical (28). seemedj 2021, vol 5, no. 1 medical vs surgical abortion 185 southeastern european medical journal, 2021; 5(1) estonia legal within the eleventh week of pregnancy (29). mifepristone in combination with misoprostol (arthrotec) for the purpose of medical abortion was registered in 2003. medical abortion can be used up to the 63rd day of pregnancy (30). finland legal within the twelfth week of pregnancy. up to 20 weeks if there is a risk to physical health of woman or if the woman is younger than 17. up to 24 weeks in case of major fetal malformation; no limit if there is the woman’s life is in danger (31). legal and free of charge, on outpatient basis. france the ten-week limit was extended to the twelfth week in 2001 (32). france was the first country to legalize the use of ru-486 as an abortifacient in 1988, allowing its use up to seven weeks of pregnancy under medical supervision. according to a united nations population division estimate, 19% of all french abortions used ru-486 as of 2002. medical abortion represents almost 50% of all performed abortions. in response to the covid-19 pandemic, france has extended access to medical abortions to nine weeks of pregnancy (33). germany legal within the first 3 months of pregnancy; mandatory counseling is required; abortion is also legal later in pregnancy in cases of medical necessity (34). legal within 9 weeks (63 days) since last menstruation, requires medical prescription (35). greece legal within the first 3 months of pregnancy (36). mifepristone and misoprostol are registered, available and affordable. however, medical prescription and hospitalization are required (37). hungary legal during the first twelve weeks of pregnancy (38). banned seemedj 2021, vol 5, no. 1 medical vs surgical abortion 186 southeastern european medical journal, 2021; 5(1) ireland legal since 2018 (following a constitutional amendment approved by a referendum in may 2018) within the twelfth gestational week and later in cases where the pregnant woman's life or health is at risk, or in the cases of a fatal fetal abnormality. abortifacient drugs are illegal (39). latvia legal during the first twelve weeks of pregnancy (40). legal and available since september 2008. prescription and gynecological assistance are required. medical abortion can also be carried out in certified in-patient facilities. lithuania legal during the first twelve weeks of pregnancy (41). banned. luxembourg legal during the first twelve weeks of pregnancy, following two consultations with a medical doctor and a psychologist, and a waiting period of at least three days (42). legal within 7 weeks (49 days) of pregnancy. malta banned under all circumstances. malta is the only country in the european union to ban abortion altogether (43). banned. the netherlands legal during the first 24 weeks of pregnancy (i.e., when it is believed that the fetus has develop vital functions enabling it to live outside of the womb) (44). legal (45). poland only legal in cases where the mother's life or health is at risk, in cases of major fetal malformations or pregnancy as a result of rape. banned (46). portugal legal during the first ten weeks of pregnancy. legal since 2007 (47). romania legal during the first fourteen weeks of pregnancy. legal with prescription (48). slovakia legal during the first twelve weeks of pregnancy. not available (49). seemedj 2021, vol 5, no. 1 medical vs surgical abortion 187 southeastern european medical journal, 2021; 5(1) slovenia legal during the first ten weeks of pregnancy. mifepristone available (50). spain legal within the 14th week of pregnancy, and at later stages in cases of serious risk to the health of the mother or fetal abnormalities. legal within the first trimester of pregnancy. not easily accessible and costly. surgical abortions account for most termination of pregnancy procedures (51). sweden legal within the twelfth week of pregnancy (52). medical abortion up to 63 days of pregnancy was approved in sweden in 1992. medical abortions accounted for 93% of all abortions in 2018 (53). united kingdom legal within 24 weeks of pregnancy (54). mifepristone, approved for use in britain in 1991, and misoprostol are legally accessible up to the ninth week (55). in italy, the legislation enacted to regulate access to voluntary termination of pregnancy (law 194/1978, titled “norme per la tutela sociale della maternità e sull’interruzione volontaria della gravidanza”, or “norms on the social protection of motherhood and the voluntary termination of pregnancy”), has effectively repealed articles 545 to 555, which used to criminalise the termination of pregnancy in any way or form (5). as a continuation of this legislative development, following the completion of the experimentation process, ru-486 has been marketed in italy since 10 december 2009. nonetheless, it is worth noting that, unlike other european countries, where it was already legal to medically terminate a pregnancy up to 63 days of amenorrhea, italy has lowered that time limit to 49 gestational days. the impact that the “procreative revolution” has had on the awareness of the population, however, has pushed the italian supreme court to issue rulings that have helped to overcome several legal and practical hurdles over the years (6, 7). materials and methods materials and study design a large and qualitative review of the literature between the seventies and 2020 has been conducted to analyse the scientific background on medical and surgical abortion. the aim was to clarify the differences among the current laws and guidelines governing voluntary termination of pregnancy in different european countries, with an eye on the italian situation, where the applicability of these laws is faced with ethical concerns. the study was conducted between 2019 and 2020 at the sapienza university of rome, department of anatomical, histological, forensic and orthopedic sciences, in collaboration with the department of medical and surgical sciences, university of foggia. methods the authors have examined the main medical databases, e.g. pubmed, google scholar, scopus and cochrane library, as well as legal databases (lexis, justia, kleagle) by applying effective combinations of terms, i.e. surgical abortion; conscientious refusal to treat; mifepristone; medical abortion; voluntary seemedj 2021, vol 5, no. 1 medical vs surgical abortion 188 southeastern european medical journal, 2021; 5(1) termination of pregnancy; abortion guidelines; health standards; emergency contraception; conscientious objection; contraception; ru-486; who guidelines on medical abortion procedures. discussion the development and availability of new procedures and treatments undoubtedly entail novel ethical quandaries, at least theoretically. the issue of procreative freedom has unfolded along two distinct and irreconcilable lines of reasoning: if, on the one hand, medically assisted procreation has made it possible for women of relatively advanced age to achieve motherhood (8, 9), voluntary termination of pregnancy is in keeping with the woman’s will not to become a mother (10). nowadays, access to abortion services, as codified in italian statutes, presents considerable difficulties, even more so in cases of unplanned pregnancies, when contraceptive methods fail or when sexual abuse results in pregnancy. in fact, access to emergency contraception, which has positively contributed to lowering abortion rates, may not be easily available in a timely fashion (11-13). contraceptive use rising as abortion falls: differences between italy and european countries in 2018, more than 64.9% of italian public hospitals guaranteed access to abortion services. hence, 35% of italian facilities fail to meet the standards set by law 194/78 (11). one of the most relevant factors that led to such a situation is certainly conscientious refusal to treat by medical personnel, which is codified as a right in law 194/78, under article 9. according to said provisions, objectors may opt out of “performing procedures and activities specifically and necessarily aimed at achieving a termination of pregnancy” (14). conscientious refusal to treat does not however exempt professionals from providing care before and after an abortion procedure or intervening in cases of emergency or imminent danger to the patient’s life. after all, the italian healthcare system is bound to uphold the free exercise of women’s right to sexual and reproductive freedom by guaranteeing access to abortion procedures through the services and professionals set in the provisions of law 194/78, by minimising the detrimental effects of conscientious refusal to treat under such a right, and possibly even ordering transfers of objecting physicians if no one else agrees to perform the procedure. the practical execution of such measures is undeniably complicated in a country such as italy, where conscientious objectors account for roughly 70% of health care professionals (15), which is an extremely high share compared to the european average – 10% in the united kingdom, 7% in france, and none in sweden (1621). in most european countries, the law allows surgical abortion upon a woman’s request in the first weeks of pregnancy or in an advanced gestational period under certain circumstances (16-51) (table 1). france was the first european country to legalise abortion by virtue of law 75-17 of 17 january 1975 (law on the termination of pregnancy). according to the french law, every pregnant woman has the right to an abortion until the twelfth week of pregnancy. after this time limit, the french law consents to abortion only if the continuation of pregnancy proves to pose a real and serious danger to the woman’s health or life (16). in france, ru-486 has been legal since 1988. the pill is administered within the first seven weeks of pregnancy and under medical supervision. in this country, medical abortions represent approximately 50% of all abortions performed. in particular, according to an estimate from the united nations population division, 19% of all abortions in france registered since 2002 have occurred by taking ru-486. moreover, in response to the covid-19 pandemic, france has extended access to medical abortion until the ninth week of pregnancy (17). in contrast, the republic of ireland has an extremely restrictive approach to abortion, seemedj 2021, vol 5, no. 1 medical vs surgical abortion 189 southeastern european medical journal, 2021; 5(1) unless there are circumstances that put a woman’s health and life at risk. in ireland, the constitution recognises the right to life for unborn children. should such conditions arise that endanger the health of the woman and/or the child, however, the law does not set time limits for terminating pregnancy, though a surgical abortion is the only possible option (47). regarding drugs used for medical abortion, there are only a few european countries where abortifacient drugs are not registered (45), or they are illegal or banned (46-51) (see table 1). in the european union, malta is the only state that has banned both surgical and medical abortion (49). these differences between various european countries, from the north to the south of europe, have historical, political and religious origins. medical abortion in spain, for example, is not easily accessible and it is very expensive. for these reasons, surgical abortions account for the majority of pregnancy termination procedures (44). conversely, in sweden, medical abortions accounted for 93% of all abortions in 2018 (19). any woman who turns to a healthcare professional has the right to thorough and comprehensive consultations on abortion practices (also, and above all, in relation to the clinical condition of a pregnant woman) as well as on the risks and benefits of one method compared to the other. consultation is advised, but not mandatory for adult women, while it is required for minors. the “contraceptive revolution”, or rather the introduction of the abortion pill, in fact, started in the 1960s and 1970s in western european countries, which were the first to legalise abortion and where, therefore, it is perceived as a fundamental right of all women. italy: government legislation and abortion plan the high degree of sensitivity in italy towards ethically and religiously contentious issues has most likely played a role in stymieing and delaying scientific progress in terms of access to abortion and medically assisted procreation (map) procedures. based on law 194 (“norms on the social protection of motherhood and the voluntary termination of pregnancy”), women may legally resort to voluntary termination of pregnancy at national public facilities within the first 90 days of gestation, after which pregnancies may only be terminated for therapeutic purposes (5). nevertheless, the share of conscientious objectors has grown by 12% over the past 10 years, reaching as much as 90% in regions such as molise, trentino-alto adige and basilicata. significantly, in the whole region of molise, there is currently only one registered physician who has not expressed a refusal to treat (11). in 2014, the european committee of social rights of the council of europe formally reprimanded three hospitals in the central marche region, jesi, fano and fermo, where all medical personnel had expressed a refusal to treat. the committee claimed that such a situation constituted a violation of women’s right to health, which is enshrined in the european social charter (52). another element negatively affects medical abortion: in compliance with the recommendations issued by the high council of health, most italian regions require women seeking abortifacient drugs, such as ru-486, to be hospitalised to terminate a pregnancy (53). due to that requirement and the organisational and ethical challenges which it engenders, many facilities have mostly opted for surgical abortion instead, to the extent that in 2018, fewer than 25% of italian women could resort to medical abortion (11). in december 2015, the association of italian physicians for contraception and abortion (amica), counting on the support of various organisations and high-profile backers, sent an open letter to the italian ministry of health, in which it asked to make medical abortion procedures less restrictive, on a day hospital basis and, where possible, accessible even in family counselling centres and ambulatory care facilities, for the sake of ensuring health care services availability and adequacy. on 8 august 2020, the ministry of health updated the set of guidelines regulating access to the abortifacient drug ru-486, allowing for its administration on an outpatient basis, i.e. with no need for seemedj 2021, vol 5, no. 1 medical vs surgical abortion 190 southeastern european medical journal, 2021; 5(1) hospitalisation, and even extending the ultimate time limit for abortion from the seventh to the ninth gestational week. such a development, based on scientific evidence, undoubtedly constitutes a meaningful step forward for italy in terms of fully enforcing law 194/78. nevertheless, such a consequential decision has occurred within an uneven socio-political context. as a matter of fact, the ministerial decision has been made on the heels of an opinion which was asked from the high institute of health, following a regulatory decision made by the regional government of umbria. that opinion was meant to discourage the tendency on the part of regions to perform medical abortion on a day hospital basis. the umbria legislative initiative followed the 2010 set of ministerial recommendations (53), after the italian medicines agency (aifa) had authorised the marketing and distribution of the drug in compliance with european standards, albeit with restrictions in place that do not apply in other countries, such as the three-day hospitalisation requirement and the seven-week gestational limit. the newly introduced ministerial directive has raised widespread controversy. the legal affairs department of the regional government of piedmont, in agreement with the italian episcopal conference, has opposed the directive, arguing that it may run counter to law 194/78 and stressing that outpatient administration of ru-486 could lead to serious complications for women. conclusion it is obviously essential that the new guidelines do not conflict with the original primary purpose of law 194/78 in terms of providing sound healthcare and psychological consultations for women in order to “try to remove the underlying reasons that induce women to seek an abortion” (5). furthermore, it is essential to guarantee access to follow-up support activities for those women who ultimately decide to terminate their pregnancies, for the purpose of ensuring the fetus is expelled in a complete and safe fashion in the interest of the patient’s well-being. furthermore, the who itself has been advocating for medical abortion (4) over surgical abortion in light of the many advantages in terms of financial benefits for healthcare providers (as a result of fewer hospitalisations, reduced use of anaesthesia and fewer surgical procedures) and a higher degree of safety, given the lower rates of complications arising from surgical interventions, such as suction aspiration and curettage. moreover, in cases where medical abortion is not carried out in a timely fashion, resorting to surgical abortion is inevitable, often as an emergency procedure, which increases the risk for patients to contract infections or haemorrhages during surgery (54, 55). the italian ministerial directive is ultimately and consistently aimed at guaranteeing and upholding the “right to responsible and conscious procreation” by removing some of the barriers hindering access to termination of pregnancy and extending the period in which such procedures can be accessed, in an effort to discourage illegal abortions and reduce the need for late surgical abortions, often performed under emergency circumstances. the nationwide application of said ministerial decree, released on 8 august 2020, will make it possible to gather more consistent and reliable data as to the rates of adverse events associated with medical abortion, which will provide scientifically reliable analytical elements for assessing its impact compared to surgical abortion procedures. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. seemedj 2021, vol 5, no. 1 medical vs surgical abortion 191 southeastern european medical journal, 2021; 5(1) references 1. world health organization. global abortion policies databases. https://abortionpolicies.srhr.org/ (date last accessed 13 august 2020). 2. perrone g. interruzione volontaria di gravidanza nei paesi dell’unione europea scheda informativa. associazione luca coscioni 2018. 3. johnson br jr, mishra v, lavelanet af, khosla r, ganatra b. a global database of abortion laws, policies, health standards and guidelines. bull who. 2017; 95: 542-4. 4. world health organization, department of reproductive health and research. safe abortion: technical and policy guidance for health systems – 2nd ed. 2012. 5. norme per la tutela sociale della maternità e sull’interruzione volontaria della gravidanza, l. no. 194, 22 maggio 1978. 6. montanari vergallo g, zaami s, bruti v, signore, f, marinelli e. how the legislation on medically assisted procreation has evolved in italy. med law. 2017; 36:5-28. 7. ricci g, delbon p, conti a, sirignano a. il difficile percorso normativo italiano sulla procreazione medicalmente assistita: revisione della letteratura e stato dell’arte [literature review and state of the art of the italian law on medically assisted reproduction]. clin ter. 2015; 166:e234-e241. 8. napoletano s, del rio a. reproductive medicine between advancement and ethics. clin ter. 2018; 169:e108-e109. 9. turner jv, mclindon la. bioethical and moral perspectives in human reproductive medicine. linacre q. 2018; 85:385-398. 10. negro f, varone mc, del rio a. advances in medically-assisted procreation technologies: can malpractice claims and "reproductive damage" be identified. clin ter 2020; 171:e225e228. 11. ministero della salute. relazione ministro salute attuazione legge 194/78 tutela sociale maternità e interruzione volontaria di gravidanza dati 2018-2020. 12. zaami s, signore f, baffa a, votino r, marinelli e, del rio a. emergency contraception: unresolved clinical, ethical and legal quandaries still linger. panminerva med. 2020; 10.23736/s0031-0808.20.03921-x. 13. signore f, baffa a, votino r. emergency contraception: are the rights to conscience and to reproductive freedom irreconcilable? clin ter. 2020; 171:e237-39. 14. montanari vergallo g, zaami s, di luca nm, marinelli e. the conscientious objection: debate on emergency contraception. clin ter. 2017; 168:e113-e119. 15. minerva f. conscientious objection in italy. j med ethics. 2015; 41:170-73. 16. loi n° 82-1172 du 31 décembre 1982 relative a la couverture des frais afferents a l'interruption volontaire de grossesse non therapeutique et aux modalites de financement de cette mesure. 17. elzas s. france extends access to abortions during covid-19 pandemic. rfi. issued on 11th april 2020. 18. abortion act 595, 14th june 1974, amended 1995:660 and 2007:998. reform promulgated on 19th may 2005, with amendments up to and including swedish code of statutes 2005:294; entered into force 1st july 2005. 19. statistik om aborter 2018". national board of health and welfare. 22nd may, 2019. retrieved 23rd may 2019. 20. abortion act, 17th october 1967, later amended via the human fertilization and embryology act, on 24th april 1990. 21. rowlands s. contraception and abortion. j r soc med 2007; 100:465-8. 22. federal law passed on 23rd january 1974. (bundesgesetzblatt, no. 60, 1974). seemedj 2021, vol 5, no. 1 medical vs surgical abortion 192 southeastern european medical journal, 2021; 5(1) 23. zusammenfassung der merkmale des arzneimittels, at-2014/03; humanarzneispezialitäten, veröffentlichung (12/2012) über zulassungen von arzneispezialitäten sowie über änderungen und aufhebungen von zulassungen. 24. law on termination of pregnancy, enacted on 3rd april 1990. national evaluation committee (law of 13 august 1990). https://www.loc.gov/law/help/abortionlegislation/abortion-legislation.pdf 25. law no. 1252-1978, passed on 21st april 1978, act concerning the medical measures for materialization of the right to freely decide on the birth of children". 26. "parliament decriminalises abortion cyprus mail". cyprus mail. retrieved 18th august 2018. https://cyprusmail.com/2018/03/30/parliamentdecriminalises-abortion/ 27. law 66 and regulation 75, 1986, effective 1 january 1987. regulation from ministry of health 467/1992. 28. legislation denominated lovtidende for kongeriget danmark, part a, 6 july 1973, no. 32, pp. 993-99, amended through law n° 389, 14 june 1995 and lbk no. 95 07/02/2008. 29. fiala c, gemzel-danielsson k. review of medical abortion using mifepristone in combination with a prostaglandin analogue. contraception 2006; 74:66-86. 30. ‘termination of pregnancy and sterilization act’ enacted on 25 november 1998 by the estonian parliament. 31. world health organization. global abortion policies database. last updated: 14th november 2018. 32. abortion act of 1970 (law 239, 24 march 1970) reformed by law n° 564, 19 july 1978 and law n° 572, 12 july 1985; law n° 328, 2001. 33. german criminal code in the version published on 13 november 1998 (federal law gazette i, p. 3322), as last amended by article 2 of the act of 19 june 2019 (federal law gazette i, p. 844) section 218a: exemption from punishment for abortion (strafgesetzbuch – stgb)218a: straflosigkeit des schwangerschaftsabbruchs. 34. bundeszentrale für gesundheitliche aufklärung. der medikamentöse schwangerschaftsabbruch. updateded on 25.09.2019. 35. law 1609/1986, enacted on 3rd july, 1986. 36. ioannidi-kapolou e. use of contraception and abortion in greece: a review. reprod health matters 2004; 12: 174-83. 37. “sexual and reproductive health law”, enacted by the parliament on 31st january 2002, entered into force on 1 july 2002, which also lays out the grounds for the termination of pregnancy. 38. law “on sexual information, illegal abortion and termination of pregnancies”, enacted on 15th november 1978, which amended penal code act 348-353. 39. termination of pregnancy act, enacted in 1981, came into force in 1984. 40. loeber oe. motivation and satisfaction with early medical vs. surgical abortion in the netherlands. reprod health matters 2010; 18: 145-53. 41. villanueva t. portugal is ready to decriminalise abortion. bmj 2007; 334: 332. 42. codul penal al romaniei (new penal code), in force since 2014. art. 201 (1) : intreruperea cursului sarcinii. 43. law on medical measures to implement the right to a free decision regarding the birth of children, 1977. 44. ley orgánica 2/2010 de salud sexual y reproductiva y de la interrupción voluntaria del embarazo, articles 13 and 14. 45. decree no. 2 of 1 february 1990 on the conditions and procedures for the artificial termination of pregnancy. seemedj 2021, vol 5, no. 1 medical vs surgical abortion 193 southeastern european medical journal, 2021; 5(1) 46. act lxxix, enacted on 17th december 1992 on ‘the protection of the life of the fetus’. latest modification: june 2000 (lxxxvii). 47. the health (regulation of termination of pregnancy) act 2018 (act no. 31 of 2018; previously bill no. 105 of 2018. 48. abortion has been regulated since 1994 by a decree of the lithuanian minister of health which replaced the former soviet law and restricted again the grounds for abortion beyond 12 weeks of pregnancy. 49. criminal code of malta (chapter 9 of the laws of malta, “to amend and consolidate the penal laws and the laws of criminal procedure”, 10th june 1854. 50. ‘law on family planning, human embryo protection and conditions of abortion’ enacted on 7th january 1993. further restrictive amendments were introduced in january 1997. 51. law 73, 23rd october 1986, effective since january 1987 and amended through law n° 419/1991. 52. european committee of social rights. decision on admissibility and the merits: confederazione generale italiana del lavoro (cgil) v. italy, complaint no. 91/2013. 53. ministero della salute. linee di indirizzo sulla interruzione volontaria di gravidanza con mifepristone e prostaglandine. issued on 16th july, 2010. 54. zaami s, montanari vergallo g, napoletano s, signore f, marinelli e. the issue of delivery room infections in the italian law. a brief comparative study with english and french jurisprudence. the journal of maternal-fetal & neonatal medicine 2017; 31:223-7. 55. miller ec, gallo m, kulick er, friedman am, elkind msv, boehme ak. infections and risk of peripartum stroke during delivery admissions. stroke 2018; 49:1129–34.. author contribution. acquisition of data: negro f; varone mc, cotoia a; beck r administrative, technical or logistic support: negro f; varone mc, cotoia a; beck r analysis and interpretation of data: negro f; varone mc, cotoia a; beck r conception and design: negro f; varone mc, cotoia a; beck r critical revision of the article for important intellectual content: negro f; varone mc, cotoia a; beck r drafting of the article: negro f; varone mc, cotoia a; beck r final approval of the article: negro f; varone mc, cotoia a; beck r seemedj 2020, vol 4, no. 2 the impact of physical activity and sports on academic achievement 97 southeastern european medical journal, 2020; 4(2) original article the impact of physical activity and sports on academic achievement of students in primary and secondary schools in osijek-baranja county, croatia 1 anja vučić *1, vesna bilić-kirin 2,3 1 healthcare centre, family medicine, vukovar-srijem county, vinkovci, croatia 2 institute of public health for the osijek-baranja county, department of medicine for school-age children, osijek, croatia 3 faculty of medicine osijek, josip juraj strossmayer university of osijek, croatia *corresponding author: anja vučić, anja_osk@hotmail.com received: jul 7, 2020; revised version accepted: oct 27, 2020; published: nov 12, 2020 keywords: physical activity, obesity, academic success, schools abstract introduction: the study aimed to examine the prevalence of sports outside of regular school classes among primary and secondary school students. the secondary aim was to study the correlation of physical activity, students’ socioeconomic status and parents’ level of education with students’ educational outcomes. material and methods: data were collected from medical records of the croatian institute for health insurance in the osijek-baranja county and through regular physical examination of students in the fifth and eighth grade of primary school and the first grade of secondary school. results: over a 5-year period, 120 boys and 141 girls were examined; 66.28% of the students were involved in sports activities in the fifth grade, 49.04% were involved in sports in the eighth grade, and in the first grade, 43.68% of students were involved in sports. no statistically significant differences were observed in the seventh and eighth grade of primary school (p = 0.076) and in the first grade of secondary school (p = 0.057). students in the seventh and eighth grade who played sports had slightly higher grades (4.45 ± 0.68) compared to those who did not participate in sports activities (4.3 ± 0.69). similar results were obtained for students who were involved in sports in the first grade of secondary school, who had achieved slightly better results in the seventh grade (4.41 ± 0.69) compared to those who did not participate in sports activities (4.41 ± 0,69). conclusion: the results of this study suggest that physical activity in children could be associated with better school performance, which may have implications for sports having positive health benefits in both childhood and adulthood. (vučić a, bilić-kirin v. the impact of physical activity and sports on academic achievement of students in primary and secondary schools in osijek-baranja county, croatia. seemedj 2020; 4(2); 97-107) seemedj 2020, vol 4, no. 2 the impact of physical activity and sports on academic achievement 98 southeastern european medical journal, 2020; 4(2) introduction the link between exercise and children’s mental functioning has not, until recently, been systematically researched (1). exercise in childhood is associated with greater cognitive control, memory and academic achievement (26,1,7-9). growing evidence suggests that cognitive and academic differences due to exercise may have an underlying cerebral biological foundation. specifically, children that are more active have greater brain structural volume in the hippocampus and dorsal striatum, two subcortical regions important for memory and learning, as well as more efficient brain activation patterns measured by magnetic resonance imaging (mri) and event-related potential (erp) during surveillance and interference tasks over lower-ability peers (36,1,7,10-12). it is believed that exercise increases both cerebral blood flow and alertness, accelerates the development of brain-derived neurotrophic factor related to neuronal growth and plasticity, as well as stimulates angiogenesis and neurogenesis in the hippocampus. besides, research has shown that there might be indications of vascularization and irregular growth in the prefrontal cortex in brain regions related to executive functions (13-17). exercise could improve other biological outcomes such as bone density, arterial wall elasticity and general mental health (18,19). despite the many benefits of physical activity and exercise, obesity in children is a growing problem in developed countries. in 2011, 31.8% of school children were found to be overweight and 7.9% of children in europe were obese (20). childhood obesity has been shown to increase the risk of chronic diseases in adulthood, such as cardiovascular disease, type 2 diabetes, certain types of cancer, and osteoarthritis (21,22). important adverse effects on growth, blood pressure, lipids, and glucose metabolism, as well as respiratory problems such as asthma and obstructive sleep apnoea have also been reported. childhood obesity is also considered to be an important risk factor for early adulthood myocardial infarction (23-27). based on the abundance of positive effects, physical activity and exercise, as well as playing sports in school, may be beneficial to children; in contrast to obesity, physical activity has a potential positive correlation with academic achievement (28,29). material and methods for this study, data were obtained from medical records of the croatian institute for health insurance in the osijek-baranja county in the period between 2013 and 2018. data were collected through regular longitudinal physical examinations conducted in the fifth and eighth grade of primary school and in the first grade of secondary school. the study sample included 120 boys and 141 girls from osijek and baranja. additional data collected included participation in sports activities throughout the fifth and eighth grade of primary school and the first grade of secondary school, academic achievement at the end of the fourth, seventh and eighth grade of primary school, form of education continued after primary school (in regard to vocational and grammar school education programmes). physical characteristics, which include body mass index (bmi) and the locomotor system, were also reported. demographic data containing socioeconomic factors, such as the place of residence, parents’ professional qualifications and their age, were likewise collected. written parental consent was obtained before conducting this study. the study was approved by the ethics committee of the institute of public health for the osijek-baranja county. statistical analysis the data were statistically analysed using the statistica software (statsoft, oklahoma, usa, version 11). categorical variables are presented descriptively, using absolute and relative frequencies, and numerical variables are presented using arithmetic mean and standard deviation. distribution was tested for normality using the kolmogorov-smirnov test. differences between categorical variables were examined seemedj 2020, vol 4, no. 2 the impact of physical activity and sports on academic achievement 99 southeastern european medical journal, 2020; 4(2) using the χ2 test, and among numerical variables, one-way and two-way repeated measurements anova was used. p-levels lower than 0.05 were considered statistically significant. results the study included 120 boys and 141 girls (p = 0.194), 59% of them living in a city and 41% living in the countryside. considering the parents’ level of education, 59% of mothers and 62.45% of fathers completed secondary education, 28.35% of mothers and 27.2% of fathers are university graduates, while 12.64% of mothers and 10.34% of fathers finished primary school. 54.02% of students enrolled in a vocational school and 45.98% went to a grammar school (table 1). table 1. general information about the respondents n (%) p gender male 120 (45.98) 0.194* female 141 (54.02) respondents’ secondary school vocational school 141 (54.02) 0.194* grammar school 120 (45.98) involved in sports 5th grade of primary school 173 (66.28) < 0.001* 8th grade of primary school 128 (49.04) 0.757* 1st grade of secondary school 114 (43.68) 0.041* academic achievement, 4th grade of primary school 4th grade of primary school 4.7 ± 0.5 < 0.001† 7th grade of primary school 4.38 ± 0.69 8th grade of primary school 4.48 ± 0.6 *χ2 test, †repeated measures anova, data are expressed and arithmetic mean and s.d. – standard deviation when observing academic achievement, the grade point average in the fourth grade of primary school was 4.7 ± 0.5, in the seventh grade it was 4.38 ± 0.69, and in the eighth grade it was 4.48 ± 0.6. in the fifth grade of primary school, 66.28% of students were involved in sports, 49.04% were involved in sports activities in the eighth grade, and 43.68% were involved in sports in the first grade of secondary school. statistically significant differences were found in bmi percentiles (p < 0.001). in the fifth grade of primary school, the average bmi percentile was 65.6 ± 28.74, in the eighth grade, it was 59.48 ± 27.55, and in the first grade of secondary school, it was 59.43 ± 28.23. an increase in the incidence of scoliosis is observed in older students. the lowest percentage of scoliosis (4.6%) was observed in the fifth grade of primary school, while twice as many eighth grade students (9.2%) had scoliosis; however, the highest number was observed in the first grade of secondary school (12.25%). in contrast, the students’ posture improved inversely. fifth and eighth grade students had an overall poor posture (20.31%), while an improvement was observed in the first grade of secondary school, where the numbers decreased (17.24%). observing the students’ feet, an increase of about 12% was observed in the proportion of seemedj 2020, vol 4, no. 2 the impact of physical activity and sports on academic achievement 100 southeastern european medical journal, 2020; 4(2) normal findings with increasing age of the students (p = 0.008). in the fifth grade of primary school, the proportion of regular findings was 60.77%, in the eighth grade it was 60.92%, and in the first grade of secondary school it was 72.03% (table 2). table 2. the physical status of the spine and feet 5th grade of primary school 8th grade of primary school 1st grade of secondary school n (%) p* physical findings of the spine proper 196 (75.1) 181 (69.35) 176 (67.43) < 0.001 kyphosis 0 (0) 3 (1.15) 8 (3.07) scoliosis 12 (4.6) 24 (9.2) 32 (12.26) poor posture 53 (20.31) 53 (20.31) 45 (17.24) normal findings, feet status 158 (60.77) 159 (60.92) 188 (72.03) 0.008 *χ2 test no statistically significant interactions were observed between academic achievement and sports. the highest achievement in all cases was in the fourth grade of primary school, followed by a decline in achievement and then an increase in the eighth grade of primary school (figure 1). statistically significant values were not recorded in the seventh grade, but were present in the eighth grade (p = 0.076) and in the first grade of secondary school (p = 0.057). students who were involved in sports in the eighth grade of primary school had a slightly higher achievement rate in the seventh grade (4.45 ± 0.68) compared to those who did not play sports (4.3 ± 0.69). similar results were found for students who played sports in the first grade of secondary school, who had a better performance in the seventh grade (4.41 ± 0.69) compared to those who did not play sports (4.41 ± 0.69) (table 3).. table 3. the correlation between academic achievement and sports achievement in the 4th grade p** achievement in the 7th grade p** achievement in the 8th grade p** p* playing sports in the 5th grade no 4.64 ± 0.53 0.112 4.32 ± 0.64 0.493 4.47 ± 0.57 0.509 0.401 yes 4.74 ± 0.48 4.4 ± 0.72 4.48 ± 0.62 playing sports in the 8th grade no 4.68 ± 0.53 0.447 4.3 ± 0.69 0.076 4.41 ± 0.62 0.452 0.16 yes 4.73 ± 0.46 4.45 ± 0.68 4.54 ± 0.57 playing sports in the 1st grade of secondary school no 4.69 ± 0.51 0.859 4.35 ± 0.69 0.057 4.45 ± 0.6 0.424 0.923 yes 4.73 ± 0.48 4.41 ± 0.69 4.51 ± 0.6 * two-way anova, * student’s t-test seemedj 2020, vol 4, no. 2 the impact of physical activity and sports on academic achievement 101 southeastern european medical journal, 2020; 4(2) figure 1. presents the relationship between gpa and sports activities at three different points in time. no statistically significant differences in interactions were found (p = 0.437). there are indications that students who were involved in sports throughout their education had slightly higher academic achievement, although the results are not statistically significant. figure 1. the relationship between gpa and sports activities at three different points in time gpa – grade point average, 0 – no sports activities, 1 – sports activities during all school years, p = 0.437 figure 2. presents gpa scores in children who were involved in sports for at least one school year. statistically significant differences were found in the interactions (p = 0.048). in the fourth grade of primary school, girls who played sports had higher academic achievement than those who did not play sports, while this difference was not statistically significant in boys. in the seventh grade of primary school, the differences were not statistically significant, and there was considerable variety in the results of children who did not play sports compared to those who did. it is similar in the eighth grade of primary school. girls who played sports had slightly better achievement than those who did not, but the results were not statistically significant. figure 2. gpa scores in children who were involved in sports for at least one school year gpa – grade point average, 0 – no sports activities, 1 – sports activities during at least one school year, p = 0.048 figure 3. impact of parents’ level of education on academic achievement of students seemedj 2020, vol 4, no. 2 the impact of physical activity and sports on academic achievement 102 southeastern european medical journal, 2020; 4(2) figure 3 contains school outcomes in different grades, based on students’ sports activities and their parents’ professional qualifications. based on the parents’ levels of education, no differences were observed in the interactions between their professional qualifications and sports activities (p = 0.173 for maternal, and p = 0.109 for paternal education). discussion the recommended daily amount of moderate to vigorous physical activity is 60 minutes (18,30). only one third of young people worldwide are found to be sufficiently active (31). a populationbased study found that more than 50% of children in australia did not meet these recommendations (32-35). according to the national health and nutrition examination survey (nhanes), only 42% of children in america participate in the recommended daily amount of physical activity (36,35), while approximately 67% of children and young people in canada do not participate in the recommended amounts of daily physical activity (37). a study from japan, however, states that, according to a 2017 national nationwide survey conducted by the national institute for educational policy research, 67.3% of third-year secondary school students participated in extracurricular sports activities, and 83.7% of such students were involved in sports for 1 hour a day or more (38). from the data collected in physical examinations in the osijek and baranja area, we could not determine how much daily activity was moderate to severe physical activity, but what we noted was that in the fifth grade of primary school, 66.28% of students were involved in sports, in the eighth grade it was 49.04% students, and in the first grade of secondary school, it was only 43.68% students. although increasing physical activity and playing sports has been suggested as a way of improving children’s health and academic achievement, there have often been contradictions between various studies regarding exercise, aerobic exercise and academic achievement. this may be due to a lack of measurement parameters if only physical activity or exercise is measured, without the inclusion of other socioeconomic variables. in this study, no statistically significant differences were observed between the interaction of academic achievements and sport. no statistically significant differences were recorded in the seventh grade, in the eighth grade and in the first grade of secondary school between children who played sports and those who did not. children who played sports in the eighth grade of primary school had slightly higher achievement in the seventh grade in comparison with those who did not play sports. similar results were found for students who were involved in sports in the first grade of secondary school, who had slightly better success in the seventh grade than those who did not play sports. results similar to ours were also obtained in the research by david m. hansen et al. (39), who studied the linear and nonlinear relationship between physical activity and aerobic exercise with children’s academic achievement. this research did not show a significant correlation between academic achievement and physical activity. another study published in 2016 also found no significant correlation between moderate to vigorous physical activity and working memory or academic achievement when comparing children involved in activity measurement and those excluded by age, gender, adhd, height, weight, bmi, puberty stage, exercise, overweight or obesity (40). however, there are many more studies that show a positive correlation between academic achievement and sports. in a study conducted in finland on children from the age of 12 (n = 1,723, 49% boys) and 15 (n = 2,445, 48% boys) until they were 40 years old, jaana t. kari et al. (41) confirmed the longitudinal correlation between physical activity and educational outcome, but also that physical activity in adolescence may not only predict academic achievement during compulsory primary education, but may likewise improve educational outcomes later in life. seemedj 2020, vol 4, no. 2 the impact of physical activity and sports on academic achievement 103 southeastern european medical journal, 2020; 4(2) in south korea, students who were more physically active were generally found to have higher academic achievement. in addition, their final exam scores in english, mathematics and science were significantly correlated with the paps (physical activity promotion system) scores (42). extensive research has also been conducted in australia to measure cardiovascular endurance, muscle strength, strength and academic achievement at school. they also found a positive correlation between physical activity and academic achievement, which is inconsistent with our results (43). the same goes for a study of the california department of education, which observed aerobic capacity, body composition, strength, and flexibility in addition to california standard tests for assessing language, the arts, and mathematics success as academic identifiers (44). other similar research on this topic was conducted by castelli et al. (8). they measured aerobic performance, flexibility, muscle strength and found that exercise had a positive effect on mathematics achievement and overall academic achievement, while stevens et al. (45) found a positive correlation of physical activity with mathematics and reading success achieved among boys and girls. sometimes different results are observed between boys and girls. in japanese adolescents, a hybrid approach reported a positive effect of exercise on boys’ academic achievement, but was observed as not statistically significant among girls (46). if we compare these same differences in our research, we can see that in the fourth grade of primary school, girls who played sports had higher academic achievement than those who did not play sports, while this difference was not statistically significant in boys. in the seventh grade of primary school, the differences were not statistically significant, and there was greater variability in the results of children who did not play sports compared to those who did. this is similarly observed in the eighth grade of primary school. girls who played sports had slightly better academic achievement than those who did not; however, the results were not statistically significant. in relation to sports and other physical activity, obesity is becoming a growing problem in school children. corresponding to this thesis, it has been investigated how weight can affect academic achievement, but the evidence itself is not conclusive. luis b. sardinha et al. (47) noted that cardiorespiratory exercise and weight were independently and collectively linked to academic performance of seventh grade students. one study found that in turkey, among children aged 10-19 in urban areas, one in five were obese; the incidence of obesity among children in urban areas was thus twice as high as in children from rural areas (48). such data indicate a significant need for research to improve our understanding of the factors contributing to the high prevalence of childhood obesity, as well as for the development of potential urban intervention strategies (49). our study involved 120 boys and 141 girls (p = 0.194), where 59% of children live in the city and 41% live in the countryside. it has been confirmed that academic success may be correlated with the parents’ level of education, as well as with socioeconomic status (41). looking at the professional qualifications of parents, we observed that 59% of mothers and 62.45% of fathers completed secondary education, 28.35% of mothers and 27.2% of fathers are university graduates, while 12.64% of mothers and 10.34% of fathers finished primary school. in our study, based on the data about education after primary school, we found that 54.02% of students enrolled in a vocational school and 45.98% in a grammar school. parents’ level of education lower than secondary school qualifications negatively affected academic achievement in boys and girls in a study conducted in japan, which observed the students’ academic success (46). conclusion today, more and more studies are being conducted on this topic and similar topics, and each of these studies highlights the positive effects of physical activity in children. seemedj 2020, vol 4, no. 2 the impact of physical activity and sports on academic achievement 104 southeastern european medical journal, 2020; 4(2) nonetheless, the results are mostly devastating in the sense that fewer and fewer children are engaged in physical activity and sports. what is even more worrying is that there has been an increase in health problems in children. although our data did not show a significant statistical correlation between sports activities and academic achievement, research alone could contribute to a better understanding of the subject, as well as to the creation of positive habits that influence the reduction of overweight and obesity, and thus also the diseases associated with it. the disadvantages of this research are that it was not possible to determine the exact amount of physical activity in children involved in sports or the intensity of exercise. since it has been proven that physical activity improves the quality of life, it is necessary to involve local communities in organising sports activities that would be easily accessible to children and parents, which would increase the number of interested children and adults. it is important to make society aware of the fact that sports and sports activities are not reserved exclusively for top athletes, but that they are an important aspect of child development and adult recreation for persons who care about their physical and mental health. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. the authors whose names are listed certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript. i references 1. tomporowski pd, davis cl, miller ph, naglieri ja. educ psychol rev. 2008; 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4 (1):1-7. 49. steenson s, ozcebe h, arslan u, unlu hk, araz om, yardim m, uner s, bilir n, huang tt-k. assessing the validity and reliabilitiy of family factors on physical activity: a case study in turkey. plos one 2018; 13(6):e0197920. doi: 10.1371/journal.pone.0197920 i author contribution. acquisition of data: vučić a, bilić-kirin v administrative, technical or logistic support: vučić a, bilić-kirin v conception and design: vučić a, bilić-kirin v critical revision of the article for important intellectual content: vučić a, bilić-kirin v drafting of the article: vučić a, bilić-kirin v final approval of the article: vučić a, bilić-kirin v guarantor of the study: vučić a, bilić-kirin v provision of study materials or patients: vučić a, bilićkirin v seemedj 2019, vol 3, no. 2 major subject of study physical activity sedentary behaviour 22 southeastern european medical journal, 2019; 3(2) original article self-determined engagement in physical activity and sedentary behaviour of college students in eastern croatia does the major subject of study make a difference?1 erna davidović cvetko 1, dražen pejić 1 1 college of applied sciences “lavoslav ružička” in vukovar, croatia corresponding author: erna davidović cvetko, erna.davidovic@vevu.hr received: sep 30, 2019; revised version accepted: dec 16, 2019; published: dec 16, 2019 keywords: physical activity, sedentary behaviour, college students abstract aim: the aim of this study was to investigate physical activity and sedentary behaviour of college students who study health sciences (physiotherapy) and students who study non-health-related majors (law and economics) at the same college to find out if there is any impact of their major subject of study on how they spend their leisure time, as well as to examine sex differences in physical activity and sedentary behaviour. methods: a cross-sectional study was conducted during the winter semester of 2017. a total of 112 students volunteered to participate in the study, of whom there were 42 males (37%) and 70 females (63%). physical activity was measured by using a short form of ipaq (international physical activity questionnaire) and sedentary behaviour by an sbq (sedentary behaviour questionnaire). additionally, some general questions were included, such as sex, body height , body weight and field of study. study participants were divided into two groups, according to their major: 1. students of physiotherapy (55 participants); and 2. students of law and economics (57 participants). results: the participants reported a high physical activity level, but also a significant amount of time spent on sedentary activities. results showed that there was no difference in physical activity levels, but that there was a slight difference in sedentary behaviour between physiotherapy students and students of law and economics with regard to the following: total time spent in sedentary activities during weekdays (p=0.006), involvement in sitting and driving/riding in a car, bus or train on weekdays, and time spent playing computer/video games on weekend days (p=0.046). conclusion: presented results lead to the conclusion that the majority of students are sufficiently active, but still spend much time on sedentary activities. students with different preferences also differ in how they spend their leisure time, but do not differ in physical activity level. (davidović cvetko e, pejić d. self-determined engagement in physical activity and sedentary behaviour of college students in eastern croatia does the major subject of study make a difference?. seemedj 2019; 3(2); 22-32) seemedj 2019, vol 3, no. 2 major subject of study physical activity sedentary behaviour 23 southeastern european medical journal, 2019; 3(2) introduction physical inactivity is the fourth leading risk factor responsible for 6% of deaths around the world, according to the world health organization (who) (1). it presents a threat to global health due to its great prevalence among all age groups and correlation with weight gain, cardiovascular diseases, diabetes and many other medical conditions (2). current international physical activity (pa) guidelines recommend that an adult must engage in at least 150 minutes of moderate-intensity aerobic activity or 75 minutes of vigorous-intensity activity (600 met-min) weekly (3). when it comes to inactivity, young adults are one of the vulnerable age groups since they are at a stage of life when their habits and lifestyle are forming. in the past decade, there has been a growing number of studies investigating physical activity and inactivity among young adults, mostly university and college students. studies have measured physical activity mostly by selfevaluation on the basis of questionnaires, with the ipaq (international physical activity questionnaire) being one of the most popular due to its good metric properties (4-10). most of the studies investigating physical activity among university and college students reported low physical activity, with a minority of them meeting physical activity guidelines (11, 12). also, sedentary behaviour is fairly widespread among young adults (13), measured by screen-time or the use of mobile phones. sedentary behaviour is also regarded as a risk factor for many diseases and it is affecting health independently of physical inactivity (14). physical inactivity should be differentiated from sedentary behaviour. people can be both highly active and highly sedentary (15). for those reasons, sedentary behaviour and physical activities should be investigated as two distinct modes of behaviour. female students are usually more inactive than male (16,17), but that does not necessarily mean that they are spending more time in sedentary activities. however, we found that there is a lack of studies investigating the difference between students from different study groups. yang and collaborators reported that a 7-week course on knowledge, attitude and practice of health behaviour improved health-related behaviour in chinese college students, including an increase in high physical activity involvement and a decrease in screen-time (18). this led to an increase in interest for examining the differences between students of health sciences and students majoring in non-health-related studies. students of health sciences should be better informed about the benefits of physical exercise and the effects of sedentary behaviour on health than the students who study non-health related majors. physical therapy students should be especially aware of physical activity benefits, as well as of adverse effects of sedentary behaviour, considering that, after graduation, their profession should include active lifestyle promotion and safeguarding of health. the main hypothesis of this paper was developed based on the aforementioned and states as follows: students of physiotherapy are more active, with higher physical activity levels, and spend less time in sedentary activities, compared to students who study law or economics at the same college. we also examined sex differences in physical activity levels and sedentary behaviour. participants and methods participants a cross-sectional study was conducted during the winter semester of 2017. all first-year students at the college of applied sciences in vukovar were invited to participate in the study. one hundred and twelve students volunteered to participate. among them, there were fortytwo males (37%) and 70 females. the participants’ age was between 18 and 22 years (median age 19). study participants were divided into two groups, according to their major subject of study: seemedj 2019, vol 3, no. 2 major subject of study physical activity sedentary behaviour 24 southeastern european medical journal, 2019; 3(2) 1. students of physiotherapy (55 participants); and 2. students of law and economics (57 participants). all participants signed informed consent forms. measures the survey included a short form of ipaq (international physical activity questionnaire) (19) and an sbq (sedentary behaviour questionnaire) (20). additionally, some demographic items were included, such as sex, body height, body weight and field of study. the short form of ipaq consists of seven items. six items measure three levels of physical activity (light (walking), moderate and vigorous) and one measures daily sitting time. according to craig and colleagues (21), ipaq is characterized by a good test-retest reliability coefficient (icc) ranging between 0.81 and 0.89 and criterion validity (spearman coefficient) ranging from 0.26 to 0.27. metabolic equivalent minutes (met min/wk) and time spent in physical activity per week were calculated by following the scoring protocol (22). time spent in physical activity (pa) per week was converted into time spent in physical activity per day (min pa/7). sbq measures time spent in 9 sedentary activities during weekdays and weekend days separately. there are 9 items that determine the amount of time spent doing 9 sedentary activities during weekdays and weekend days: watching television, playing computer/video games, sitting while listening to music, sitting and talking on the phone, doing paperwork or office work, sitting and reading, playing a musical instrument, doing arts and crafts, sitting and driving/riding in a car, bus, or train. according to rosenberg and colleagues (23), the iccs were acceptable for all items and the total scale (range=0.51-0.93). for men, there were significant relationships of sbq items with ipaq sitting time and bmi. for women, there were relationships between the sbq and accelerometer inactivity minutes, ipaq sitting time, and bmi. the participants gave their answers by selecting the amount of time they spend performing the relevant sedentary activity on a typical weekday in the first part, and on a typical weekend day in the second part of the sbq (from when they wake up until they go to bed). frequencies of answers were compared between the tested groups. bmi (body mass index) was calculated based on self-reported height and weight. statistical analysis statistical analysis was performed using ibm spss statistics 20. numerical variables: bmi value, time spent in sitting, walking, and in moderate and vigorous activities were tested for normal distribution. normally distributed data were presented as a mean and standard deviation, while nonnormally distributed data were presented as median and range. the difference between study types (majors) was tested by applying the mann-whitney u-test for independent samples. it was used to compare ipaq results. the difference in frequency of answers in the sbq was tested by using the chi-square test, as well as the difference in frequency between physical activity levels according to ipaq score, and the difference in frequency between bmi categories. level of significance was set at p=0.05. results total engagement in physical activities reported by the participants was from 0 to 630min/day, with the median being 120min/day, and mode being 60min/day. there was no statistically significant difference between physiotherapy students and students of law and economics (p=0.183), or between male and female students (p=0.204). table 1. shows the reported physical activity engagement levels during one typical day for physiotherapy students and for students of law and economics. seemedj 2019, vol 3, no. 2 major subject of study physical activity sedentary behaviour 25 southeastern european medical journal, 2019; 3(2) table 1. difference in time (min/day) spent in physical activities between physiotherapy students and students of social sciences type of physical activity time spent in physical activity (min/day) physiotherapy students (n=55) students of social sciences (n=57) p* min max. median mean min max. median mean vigorous pa 0 120 20 24.4 0 200 30 45.2 0.161 moderate pa 0 180 30 40 0 300 30 30 0.061 walking 0 420 30 78 0 240 60 84 0.707 * statistical significance of difference derived from the mann-whitney u-test the reported sitting time (ipaq) in a typical day is between min. 30min and max. 720min, with the median being 360min, and mode being 300min. there was no statistical difference between males and females (p=0.29), but the difference between physiotherapy students and students od law and economics was statistically significant, indicating that physiotherapy students spend more time in sitting activities in comparison to students of law and economics (p<0.0001). physiotherapy students reported between 120 and 720min of sitting per day, with median value being 420min/day, while the students of law and economics reported that they sit between 30 and 600min/day, with median value being 300min/day. reported physical activity converted to metmin/week yielded results presented in table 2. physical activity of 74% participants (83 of 112 participants) was high, with their met-min/week being over 3000 (68% of physiotherapy students and 81% of students of social sciences), while 6 (5%) participants reported physical activity lower than 600 met-min/week, which represents low physical activity (7% of physiotherapy students, and 3% of students of law and economics). pearson chi-square revealed no significant difference between physiotherapy students and students of law and economics in terms of physical activity levels (p=0.345).. table 2. difference in physical activity level expressed in met-min/week between physiotherapy students and students of social sciences type of physical activity met-min/week physiotherapy students (n=55) students of social sciences (n=57) p* min max. median mean min max. median mean vigorous pa 0 960 160 195.5 0 1600 240 362.1 0.161 moderate pa 0 1440 200 280 0 2400 280 521 0.061 walking 0 1386 198 287 0 792 198 253 0.707 total 0 2994 677 743 0 4330 640 1105 0.202 * statistical significance of difference derived from the mann-whitney u-test. seemedj 2019, vol 3, no. 2 major subject of study physical activity sedentary behaviour 26 southeastern european medical journal, 2019; 3(2) the sbq results revealed that participants were involved in sedentary activities between 60 and 720min/day on weekdays, with median value being 300min/day, and between 15 and 720 min/day on weekend days, with median value being 300min/day. there was no statistically significant difference between males and females in terms of total time spent engaging in sedentary activities during weekdays (p=0.591) or weekend days (p=0.788). a statistically significant difference was noted between physiotherapy students and students of law and economics in terms of their sedentary behaviour during weekdays, as could be seen in table 3.. table 3. difference in total time spent in sedentary activities (min/day) between physiotherapy students and students of social sciences during weekdays and weekend days time spent in sedentary activities (min/day) physiotherapy students (n=55) students of social sciences (n=57) p* min max. median mean min max. median mean weekdays 60 720 240 246 60 600 300 340 0.006 weekend days 30 600 300 300.5 15 720 330 332.5 0.372 * statistical significance of difference derived from the mann-whitney u-test. students of social sciences are more involved in sedentary activities during weekdays. the total time spent on engaging in sedentary activities is a sum of all reported times spent on engaging in sedentary activities. all those activities, along with the distribution of reported times spent in each activity, are presented for students with different majors in table 4., as are the results of the pearson chi-square test used to determine differences among groups. seemedj 2019, vol 3, no. 2 major subject of study physical activity sedentary behaviour 27 southeastern european medical journal, 2019; 3(2) table 4. difference in frequency of answers on time spent in each sedentary activity/day between physiotherapy students and students of social sciences (p = statistical significance derived from the chi-square test). 0 min /da y up to 15 min /da y 15-30 min/d ay 3060 min /da y 1-2 hours /day 2-3 hours /day 3-4 hours/ day 4-5 hours /day 6 or more hour s/da y p w e e k d a y s watching tv physioth (n=55) 13 8 1 17 10 3 2 1 0.2 38 soc sci (n=57) 7 5 9 15 13 4 2 2 playing computer /video games physioth (n=55) 27 3 7 10 6 2 0.3 79 soc sci (n=57) 29 7 6 6 4 1 1 3 sitting while listening to music physioth (n=55) 1 10 4 20 5 6 4 1 4 0.2 57 soc sci (n=57) 3 8 14 11 6 5 2 8 using phone physioth (n=55) 1 9 3 12 10 6 4 4 6 0.6 57 soc sci (n=57) 5 8 5 10 8 6 2 7 6 doing paperwor k or office work physioth (n=55) 3 6 3 18 18 4 1 2 0.1 74 soc sci (n=57) 7 8 11 15 13 1 1 1 sitting and reading physioth (n=55) 22 13 6 7 6 1 0.7 42 soc sci (n=57) 23 14 5 11 1 1 1 1 playing a musical instrumen t physioth (n=55) 49 3 2 1 0.2 21 soc sci (n=57) 55 1 1 doing arts and crafts physioth (n=55) 26 6 2 11 9 1 0.0 95 soc sci (n=57) 28 2 7 5 9 5 1 sitting and driving/ri ding in a car, bus, or train physioth (n=55) 13 8 13 13 8 0.0 09 soc sci (n=57) 2 5 7 26 12 2 2 1 w e e k e n d d a y s watching tv physioth (n=55) 8 6 5 10 12 7 4 1 0.98 3 soc sci (n=57) 7 4 6 15 13 7 3 1 playing computer /video games physioth (n=55) 29 4 3 4 7 6 1 0.52 2 soc sci (n=57) 28 5 5 9 5 4 0 2 seemedj 2019, vol 3, no. 2 major subject of study physical activity sedentary behaviour 28 southeastern european medical journal, 2019; 3(2) the difference between physiotherapy students and students of law and economics was statistically significant in terms of sitting and driving/riding in a car, bus, or train during weekdays, and in terms of sitting while listening to music on weekend days. physiotherapy students reported a maximum of 1-2 hours/day spent sitting and driving/riding in a car, bus, or train during weekdays, while some students of law and economics reported that they travel by car, bus or train for over 2-3 or more hours/day. students of law and economics also reported longer times spent sitting while listening to music on weekend days in comparison to physiotherapy students. there is a greater difference in distribution of time spent in sedentary activities between sexes. females spend more time in sedentary activities: using the phone (p=0.041 for weekend days, and p=0.009 for weekdays), doing paperwork or office work (p=0.046 for weekend days and p=0.004 for weekdays) and sitting and reading (p=0.003 for weekend days and p=0.045 for week days), while males spend more time playing computer/video games compared to females both during weekdays (p<0.001) and weekend days (p<0.001). also, females reported spending more time in doing arts and crafts on weekdays in comparison to males (p=0.036). bmi ranged between 17 and 35kg/m2, with mean value being 22.4±3.13kg/m2. most participants (86 of them, or 77%) were of normal weight (bmi from 18.5 to 24.9kg/m2), 7 (6%) were underweight, and 19 (17%) were overweight, with 4 overweight participants being in the obese category (bmi>30kg/m2). there was no continued 0 min/ day up to 15 min/ day 15-30 min/d ay 3060 min/ day 1-2 hours/ day 2-3 hours/ day 3-4 hours/d ay 4-5 hours/ day 6 or more hours /day p sitting while listening to music physioth (n=55) 5 4 5 14 9 2 5 3 6 0.04 6 soc sci (n=57) 1 4 10 16 7 5 1 13 using phone physioth (n=55) 2 10 5 10 8 7 2 2 8 0.19 3 soc sci (n=57) 7 3 8 10 7 7 5 6 5 doing paperwor k or office work physioth (n=55) 8 8 6 15 11 5 0 1 0.12 1 soc sci (n=57) 22 3 6 15 8 2 1 1 sitting and reading physioth (n=55) 26 8 5 9 3 2 1 0.93 5 soc sci (n=57) 25 9 9 7 3 2 1 1 playing a musical instrumen t physioth (n=55) 49 2 3 0.11 9 soc sci (n=57) 56 1 1 doing arts and crafts physioth (n=55) 23 1 7 5 14 3 1 0.43 8 soc sci (n=57) 28 0 5 11 8 3 2 1 sitting and driving/ri ding in a car, bus, or train physioth (n=55) 11 7 14 14 5 2 1 0.26 2 soc sci (n=57) 6 8 8 17 11 5 2 1 seemedj 2019, vol 3, no. 2 major subject of study physical activity sedentary behaviour 29 southeastern european medical journal, 2019; 3(2) statistical difference between physiotherapy students and social sciences students (p=0.073) in terms of frequency distribution by their bmi categories. discussion the main finding of this study was that there was no difference in the physical activity level, but there was a slight difference in sedentary behaviour between physiotherapy students and students of law and economics. participants reported high physical activity levels, but also a significant amount of time spent in sedentary activities. most students reported high physical activity, while only 5% of them reported low physical activity (<600 met-min/wk) and did not meet the physical activity recommendations. studies with similar objectives presented various results. some reported low physical activity levels and great amounts of time spent in sedentary activities (11-13, 24-26), while others reported results similar to ours with high prevalence of sufficiently active students (15, 27-29). the reasons for such discrepancies are still to be determined. differences in research methodology could be affecting the results and could be one of the reasons for occurrence of such discrepancies. smetaniuk and co-workers (30) identified the main themes related to the facilitators of and barriers to physical activity and sedentary behaviour in master of physical therapy students: 1) priorities and life balance; 2) commitment and accountability; 3) environment; and 4) master of physical therapy programming. variations in results pertaining to physical activity levels of participants in different studies in different places probably could be partially explained by differences in the aforementioned facilitators and barriers. in their study conducted among college students from the mid-atlantic region, us, vainshelboim and cooperatives (29) reported results similar to ours. they reported that 84-94% of examined students met the physical activity recommendations. another similarity pertains to sedentary behaviour. our results showed that approximately half of the questioned students spend 6 hours/day in sedentary activities, while they reported that 69% females and 46% males are highly sedentary. those individuals who are highly active and highly sedentary at the same time are known as “active couch potatoes” (31). they regularly exercise and meet the physical activity recommendations, but still spend great amounts of time in sedentary activities. barkley and lepp (32) reported that cell phone use is a significant positive predictor of being an “active couch potato”. mobile phones use was shown to be associated with sedentary behaviour of college students (32). however, penglee and associates examined the correlation of smartphone use and physical activity among college students in the united states and in thailand (33), and reported that greater smartphone use per day is inversely related to days per week of engaging in physical activity among thai students, but not among us students. it is common belief that sedentary behaviour is displacing physical activity, so when sedentary behaviour increases, physical activity should decrease. however, this does not have to be the case when it comes to mobile phone use. there are some smartphone functions and applications that can encourage physical activity. there are many ways in which mobile phones can be used, and if someone is a “heavy” user, their use does not have to necessarily represent time spent in sedentary behaviour. in our study, only 6 participants reported not using phones during weekdays, and 9 reported not using them on weekend days, while others use phones during the greater part of the day (table 4). we also found that there was no difference between males and females in terms of total time spent in sedentary behaviour. however, there was a difference between sexes regarding the distribution of that total time in terms of time spent on different sedentary activities. this is probably a consequence of sex differences in terms of preferences and interests. according to the review of castro et al. (34), these results differ from the results of other studies. they found that sex has no association with total sedentary time, with screen-time, or with preferences to any type of sedentary activity. we ascertained that females use phones more than males, they also spend more time doing paperwork or office work and sitting and reading, as well as doing seemedj 2019, vol 3, no. 2 major subject of study physical activity sedentary behaviour 30 southeastern european medical journal, 2019; 3(2) arts and crafts on weekdays, while males spend more time playing computer/video games compared to females, both during weekdays and weekend days. however, there is no difference between males and females in terms of total time spent in sedentary activities. as for the differences regarding sedentary activities between physiotherapy students and students of law and economics, we ascertained a significant difference in the total amount of time spent in sedentary activities during weekdays, but no difference in the total amount of time spent in sedentary activities during weekend days. during weekdays, students of law and economics reported more time spent performing the listed sedentary activities compared to physiotherapy students, even though, in the ipaq, the physiotherapy students reported more time spent sitting. this discrepancy in the results could stem from the differences pertaining to class schedule, because sitting in a classroom was not listed as sedentary behaviour in the sbq. doing paperwork and office work was included, but that could easily be understood for doing homework, preparing for class or performing other activities at home, after classes. physiotherapy students are more oriented towards practical lessons that require their involvement. this means that they do not always sit and listen during their classes, but are required to move and be physically active, unlike students who study law and economics, where classes, in general, require the students to sit and listen. in addition, other variables could also affect the differences pertaining to total time spent in sedentary behaviour during weekdays and weekend days, e.g. the distance between a student’s residence and college, which could affect the total time that a student spent sitting while driving to class. weight status of the participants was mostly within normal weight thresholds (bmi between 18.5 and 24.99kg/m2), but every fifth participant was overweight. physical inactivity is strongly correlated to bmi, and regular exercise and an active lifestyle are the best way to reduce weight and reach a healthy weight status. there were no differences between physiotherapy students and students of law and economics in terms of distribution across weight categories. young adults in general should be more educated about the risk of health problems connected to high bmi. the limitations of the study include the use of subjective measures, although valid and reliable. although researchers tried to explain the questions to the participants and help them understand and answer them as accurately as they can, the answers are still a subjective evaluation and a memory of past events, as they remember them. objective measures of physical activity and sedentary behaviour are warranted for future studies. also, the crosssectional design of the study could represent a limitation due to the inability to track changes in physical activity and sedentary behaviour, as well as their interaction, or to investigate potential facilitators and barriers that affect physical activity and/or sedentary behaviour. of course, we must take into consideration the limitation of use of ipaq. ipaq is a questionnaire that requires participants to record only physical activities (vigorous, moderate and walking) that lasted at least 10 minutes at a time. in our opinion, this is the reason why the results included 0min of walking. this result is not within the 5% of extremes, because all participants who live near the college and walk, e.g., 5min to get to their classes, or those who drive to college by car or bus and do not walk around for more than 10min do not record this brief amount of time that they spent walking. on the other hand, some students work while studying and they spend a great amount of time walking to their workplace. all presented results lead to the conclusion that the majority of students are sufficiently active, but they still spend much time in sedentary activities. our hypothesis was partially disproved, because we found no significant difference in physical activity between physiotherapy students and students of law and economics. on the other hand, physiotherapy students reported more time spent sitting, while students of social sciences reported more time spent in sedentary behaviour during weekdays. this could lead to the conclusion that, in this seemedj 2019, vol 3, no. 2 major subject of study physical activity sedentary behaviour 31 southeastern european medical journal, 2019; 3(2) case, a major affects what kind of lifestyle the students adopt and in which leisure activities they engage in. students with different preferences also differ in terms of how they spend their leisure time. in short, the student population should be provided a better education regarding the effects of sedentary behaviour on their health and wellbeing. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. . references 1. world health organization. global health risks: mortality and burden of disease attributable to selected major risks. geneva, switzerland: who; 2009. 2. gaetano a. relationship between physical inactivity and effects on individual health status. j physical education sport. 2016; 16(2): 1069-1074. 3. world health organization. global recommendations on physical activity for health. geneva, switzerland: who; 2010. 4. ballard m, gray m, reilly j, 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s, fitzgerald p, wisniewski ks. sedentary behavior and physiological health determinants in male and female students. physiol behav 2019; 204:277-282. 30. smetaniuk t, johnson d, creurer j, block k, schlegel m, burcher s, oosman sn. physical activity and sedentary behavior of master of physical therapy students: an exploratory study of facilitators and barriers. physiotherapy canada 2017; 69(3):260-270. 31. owen n, healy gn, matthews ce. dunstan dw. too much sitting: the population health science of sedentary behavior. exerc sport sci rev 2010; 38:105-113. 32. barkley je, lepp a, salehi-esfahami s. college students mobile telephone use is positively associated with sedentary behavior. am j lifestyle med. 2015; 10(6):437-441. 33. penglee n, christiana rn, battista ra, rosenberg e. smartphone use and physical activity among college students in health science related majors in the united states and thailand. int j of environ res public health 2019; 16:1315. 34. castro o, bennie j, vergeer i, bosselut g, biddle sjh. correlates of sedentary behavior in university students: a systematic review. prev med 2018; 116:194-202. seemedj 2020, vol 4, no. 1 restless legs syndrome and iron 55 southeastern european medical journal, 2020; 4(1) review article restless legs syndrome and iron 1 josipa pulić* 1 1 institute of emergency medicine of koprivnicakriževci county, koprivnica, croatia *corresponding author: josipa pulić, pulic.josipa@gmail.com introduction restless legs syndrome (rls) has been described as a neurological disorder, related to uncontrolled leg movements, and, in much received: feb 21, 2020; revised version accepted: mar 18, 2020; published: apr 27, 2020 keywords: restless legs syndrome, willis-ekbom disease, iron metabolism, serum ferritin levels, treatment smaller number of patients, it is associated with uncontrolled hand movements. uncontrolled limb movements are preceded by a sense of discomfort, described by patients as annealing, burning, tickling, etc. it occurs more frequently abstract restless legs syndrome (rls), also known as willis-ekbom disease, is one of the most common neurological disorders that significantly affects quality of life and sleep. it manifests itself in involuntary movements of the lower limbs due to the feeling of discomfort and restlessness that patients feel in their lower limbs. rls is of great interest to the experts in various fields of medicine, especially to neurologists, general practitioners, internists and psychiatrists. numerous clinical conditions and diseases play a role in the pathophysiology of rls. some of them are pregnancy, some kidney and stomach diseases, iron deficiency and some disorders of the metabolism. moreover, iron is a very important micronutrient in the human body. it is involved in many metabolic processes and, in addition to rls, it is also associated with other diseases such as hemochromatosis and anemia. this neurological disorder has wide therapeutic choices, which include lifestyle changes, dopaminergic agonists, opioids and iron therapy. many non-anemic patients with rls showed reduced levels in brain iron levels compared to healthy control groups in several research. the best course of treatment for this group of patients is iron supplementation. oral iron supplementations are the first choice of therapy for patients with low serum ferritin levels. however, when serum ferritin levels are normal or high or when oral iron is not tolerated, intravenous iron is a better choice. there are many intravenous iron formulations, but low molecular weight dextran and ferric carboxymaltose have very efficient effects on the treatment of rls. (pulić * j. restless legs syndrome and iron. seemedj 2020; 4(1); 55-62) seemedj 2020, vol 4, no. 1 restless legs syndrome and iron 56 southeastern european medical journal, 2020; 4(1) during inaction. therefore, it is a common cause of sleep disorders (1-3). in the 17th century, sir thomas willis noticed the connection between sleep disorders and lower extremity discomfort among his patients. however, karlaxel ekbom first used the term 'restless legs syndrome' in 1945. that is why this disorder is also known as willis-ekbom disease (4). unlike some other disorders with similar problems, such as polyneuropathy, the symptoms of rls decrease with leg movements. in most cases, it is a chronic disorder with worsening symptoms, which vary in intensity and frequency. therefore, in patients with chronic form of this disorder, symptoms occur at least twice a week if patients do not follow their therapy (1, 5). rls has prevalence between 8 and 10% (6) in adult and 2% in children population (7). the study conducted by manconi et al. has shown that rls occurs more frequently in women, age 35 and above, than in men of the same age (8). this disorder can be inherited or be a result of a clinical condition such as kidney diseases (hemodialysis patients) (9), pregnancy (evaluated progesterone and estrogen levels, iron deficiency) (10), anemia, stomach damage, etc. in addition, some substances such as neuroleptic drugs, caffeine, lithium, metoclopramide, antihistamines, dopaminergic agents (1, 11) may increase the risk of developing rls symptoms (figure 1). figure 1. non-hereditary causes of restless legs syndrome summarized findings from studies to date on the most common diseases/clinical conditions/drugs that are associated with restless legs syndrome. treatment for rls depends on its cause. therefore, the treatment can include dopaminergic agents, lifestyle changes, iron therapy, α-2-δligands, opioids, etc (1). compared to the previous european federation of neurological societies (efns) guidelines on the management of rls from 2004, new guidelines from 2012 bring news. numerous drug studies have been made during this period and new treatments have been examined that could be a potential therapy for rls. the majority of research deals with dopaminergic kidney diseases (hemodiallysis patients) pregnancy (iron deficiency, elevated progesteron and estrogen levels) anemiastomach damage drugs (caffeine, classical neuroleptic drugs, lithium, metoclopramide, antihistamines, treatment with dopaminergic agents) seemedj 2020, vol 4, no. 1 restless legs syndrome and iron 57 southeastern european medical journal, 2020; 4(1) agents, considered the first line therapy. the crucial role of iron in pathophysiology of rls has also been confirmed. alternative forms of therapy have been investigated; folate, vitamin e, physiotherapy, aerobic training and magnesium. nevertheless, there is still not enough evidence of their effectiveness (12). iron in relation to health and diseases iron is one of the most important micronutrient in human body. it has many functions such as a role in metabolic processes; it can cause oxidative stress because it participates in the formation of oxygen radicals (13), and it is a cofactor of numerous enzymes (13, 14). there are about 4,2 grams of iron in the human body, and much of it is bound to hemoglobin and involved in oxygen transfer (12). about 10% of the iron ingested through food is absorbed in the digestive system, mostly in the duodenum (15). ferritin is an intracellular protein that stores iron and plays an important role in regulating iron homeostasis, while transferrin is a glycoprotein that binds iron and transports it into the cells (16). the major regulator of iron homeostasis is hepcidin, 25-amino acid peptide hormone, which is mainly secreted by hepatocytes (17, 18). its deregulation is linked with excess iron and iron deficiency. therefore, when level of hepcidin is very high, like in case of inflammation, the absorption of iron is reduced and this can lead to the development of anemia (17-19). it is proved that brain and liver contain high levels of iron (13). the highest concentration of brain iron is found in substantia nigra, globus pallidus, red nucleus, putamen and dentate nucleus of the cerebellum (20, 21). in oligodendrocytes, the presence of the transferrin has been confirmed, but a greater amount of brain iron is nevertheless related with ferritin (13, 22). iron plays a large role in neurotransmitter synthesis and mitochondrial respiration and its status is regulated at the level of the bloodbrain barrier (bbb) (13, 23, 24). it has long been known that iron excess causes hemochromatosis, characterized by skin changes, weakness, loss of sex drive, abdominal pain and symptoms of diabetes; but recent studies have associated brain iron excess with the onset of neurodegenerative diseases such as parkinson's disease. on the other hand, iron deficiency in the central nervous system (cns) is associated with irritability, concentration disorder, tiredness and it may play a role in the pathophysiology of rls (13). correlation between restless legs syndrome and iron the first person who noticed that low serum iron level could be a risk factor for developing restless legs syndrome was nordlander (25, 26). ekbom observed iron deficiency among his patients. although he noticed repeated occurrence of low iron level in rls, most of his patients suffered from uremia, anemia or were pregnant women (27). in another research, o'keeffe found low serum iron and ferritin values in patients who complained of rls symptoms. it was observed that the lower the ferritin level were, the more severe rls symptoms appeared (28, 29). iron supplementation has caused improvement in some patients. the potential cause of this is altered management of brain iron in patients with rls (30, 31). two studies have shown that almost 2/5 of the patients suffering from iron deficiency anemia also had symptoms of rls. nevertheless, these studies used small groups of participants (32, 33). however, one study, conducted by allen et al. among the general population, has shown that the percentage of people with symptoms of rls is several times higher in the group of participants with iron deficiency anemia than in the general population (34). abnormalities in the concentrations of ferritin, transferrin in the cerebrospinal fluid (csf), low csf ferritin, and high csf transferrin levels have been noticed (35). circadian pattern is characteristic for rls, with symptoms being dominant at nighttime. serum iron has a circadian variation, with 30 to 50% drop at night. this can lead to clinically significant seemedj 2020, vol 4, no. 1 restless legs syndrome and iron 58 southeastern european medical journal, 2020; 4(1) drop in brain iron levels with patients with rls and create the symptoms (36). lower iron concentration in substantia nigra and putamen were found in some patients with idiopathic rls and capillary transport of iron in the brain probably plays a major role in this. in addition, it has been observed that the iron levels in substantia nigra increase with aging (3739). there is a small number of studies that have compared the connection between serum hepcidin levels and rls. however, one of them found higher prohepcidin (inactive form of hepcidin) in putamen and substantia nigra in patients with rls. this opens up the possibility of discovering new medications, such as hepcidin antagonists, for the treatment of rls (40-42). iron treatment iron deficiency anemia is present in more than 1/5 of the patients with rls (34). accordingly, oral and intravenous iron supplements are used as therapy for those patients. when serum ferritin level is lower than 75 μg/l, oral iron supplementation is the therapy of choice. while in patients with serum ferritin level higher than 300 μg/l intravenous iron preparations are a better choice (43, 44). o'keeffe observed among his patients, who had different serum ferritin values, that oral iron supplements had better effect on patients with lower serum ferritin values. however, the problem with this study was that it did not have a control group (25, 29). furthermore, oral iron supplements have almost the same effect whether taken once a day or divided into two doses. in addition, in both cases these supplementations should be taken with vitamin c in order to improve absorption in the small intestine (44, 45). according to the american academy of sleep medicine (aasm) guidelines, iron treatment is effective for rls only in patients who have low ferritin levels. in addition, it is preferred oral over parenteral iron formulations, because parenteral forms are associated with a number of side effects that can endanger patients’ life and health (46). on the other hand, intravenous iron supplementations bypass the intestinalblood barrier and restriction of iron absorption (47). intravenous iron forms take precedence over oral iron only in two cases. first is when the patient is severely bleeding and rapidly losing iron, and the second case is when patients have problems with oral iron absorption (44, 48). there are several intravenous iron formulations available: ferric carboxymaltose, iron sucrose, iron gluconate, low and high molecular weight dextrans (lmw and hmw dextrans), ferumoxytol and iron isomaltose (44). lmw dextran and ferric carboxymaltose have the best clinical evidences for treatment of rls (1, 44). infusion of 1000 mg lmw dextran improves the health of rls patients with early symptoms and significantly increases iron levels in substantia nigra (49). both oral and intravenous iron preparations have numerous limitations in the treatment of rls. the most dangerous side effect is related to hmw dextran and it involves anaphylactic shock. on the other hand, ferric carboxymaltose is safe to use because its side effects, such as nausea and headache, are much milder (50). there are not enough studies on intravenous sucrose, as well as on most intravenous iron preparations. nevertheless, it is known that intravenous sucrose is not effective for patients that do not have anemia (50). in addition, for iron gluconate, ferumoxytol and iron isomaltose, there are insufficient clinical evidences (44, 51) (table 1).. seemedj 2020, vol 4, no. 1 restless legs syndrome and iron 59 southeastern european medical journal, 2020; 4(1) table 1. iron preparation limitations in the treatment of restless legs syndrome oral ferrous sulfate: gastrointestinal upset; not effective for patients with serum ferritin level>75 μg/l ferric carboxymaltose: headache nausea iron sucrose: not effective in iron deficiency patients without anemia high molecular weight dextran: high incidence of anaphylactic shock low molecular weight dextran: not so effective in patients with symptoms of late onset rls iron isomaltose, ferumoxytol, iron gluconate: not enough clinical evidences note: summarized findings from studies to date on oral and intravenous iron preparations for the treatment of restless legs syndrome. avni et al. have proven in their research that iron preparations are safe and effective for rls. however, there is still a great need for further research, especially for the research that could determine the exact drug dosages and therapeutic regimen (43) conclusion numerous studies on the relationship between rls and iron in the body have provided better insight into the pathophysiology of this disorder and have opened up new possibilities related to therapeutic approaches, such as hepcidin antagonists. nevertheless, there are still many uncertainties related to iron therapy and a need for further research on that topic. the reason is the fact that many intravenous iron formulations lack sufficient clinical evidences regarding their effect on reducing the symptoms of rls. furthermore, many studies have a small or inadequate sample of participants. in addition, for many intravenous iron formulations the exact dosage required for the treatment of rls has not been determined.. . acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. seemedj 2020, vol 4, no. 1 stigmatization of patients with viral infections and mass psychogenic illness 60 southeastern european medical journal, 2020; 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4(1) original article cytogenetic findings in patients with intellectual disability/mental retardation and dysmorphic features in eastern croatia 1 višnja tomac* 1, 2, silvija pušeljić 1, 2, jasenka wagner 3, martina kos 1, 2, nikolina arambašić 1, 2, mia damašek 1 1 paediatric clinic, clinical hospital centre osijek, croatia 2 department of paediatrics, faculty of medicine, josip juraj strossmayer university of osijek, croatia 3 medical genetics laboratory, department of medical biology and genetics, faculty of medicine, josip juraj strossmayer university of osijek, croatia *corresponding author: višnja tomac, visnja.tomac@yahoo.com received: feb 24, 2020; revised version accepted: apr 3, 2020; published: apr 27, 2020 keywords: intellectual disability/mental retardation, chromosomal aberrations, cytogenetic, children abstract introduction: numerical and structural chromosomal aberrations are some of the most common causes of intellectual disability/mental retardation (id/mr), especially syndromic, and they represent about 10% of id/mr that can be detected using cytogenetic methods. aim: the aim of this study is to show the results of cytogenetic findings in 340 patients with id/mr and dysmorphia and/or multiple malformations in eastern croatia, examined at the paediatric clinic of the clinical hospital centre osijek and the medical genetics laboratory at the faculty of medicine osijek. methods: cytogenetic analysis of 340 samples from patients with id/mr and/or dysmorphia was conducted using g-banding with trypsin/giemsa (gtg) and fluorescent in situ hybridization (fish). results: a total of 340 patients with id/mr with dysmorphia and/or multiple malformations were referred for cytogenetic evaluation. the age range of patients was 0-18 years. the analysis included 221 boys (65%) and 119 girls (35%). a chromosomal aberration was found in 24.5% of patients. numerical aberrations (aneuploidy) were seen in 64 patients (18.8%). the most common type of autosomal aneuploidy was trisomy 21, found in 14.7% of patients. sex chromosome aneuploidy was detected in 2.6% of patients. structural abnormalities were found in 6.5% of patients. conclusion: the results of our study show that cytogenetic analysis in patients with id/mr should nowadays be applied when aneuploidies are suspected, since the first-line genetic test for patients with id/mr, especially non-syndromic, is the array comparative genomic hybridization (acgh). (tomac* v, pušeljić s, wagner j, kos m, arambašić n, damašek m. cytogenetic findings in patients with intellectual disability/mental retardation and dysmorphic features in eastern croatia. seemedj 2020; 4(1); 87-95) seemedj 2020, vol 4, no. 1 cytogenetic findings intellectual disabilitydysmorphic features 88 southeastern european medical journal, 2020; 4(1) introduction intellectual disability/mental retardation (id/mr) is defined as disability characterized by significant limitations in intellectual functioning and adaptive behaviour, covering everyday social and practical skills and starting before the age of 18 (1). it can be syndromic and nonsyndromic. the worldwide prevalence of id/mr is about 2.3% (2). genetic causes of id/mr are considered to account for 25-50% of cases (3). numerical and structural chromosomal anomalies are some of the most common causes of id/mr, especially syndromic, and they represent about 10% of id/mr that can be detected with conventional cytogenetic methods (4). major autosomal and sex chromosome aberrations often cause a number of phenotypic features, such as cardiac anomalies, infertility and growth deficiency (5). in medical genetics, cytogenetic analysis is an important source for evaluation of specific birth defects, genetic disorders, developmental delay and id/mr (6). around 1,000 chromosomal disorders have been reported (7). id/mr is one of the reasons for the referral of patients and families to genetic counselling. identification of the causes of syndromic and non-syndromic id/mr in a patient is very important because of the consequences it has for the prognosis, risk of occurrence in other family members, and prenatal diagnosis. here we will summarize the result of a cytogenetic study performed on 340 patients with id/mr and dysmorphia and/or multiple malformations in eastern croatia, who were referred to the paediatric clinic of the clinical hospital centre osijek and the medical genetics laboratory at the faculty of medicine osijek. materials and methods in this retrospective study we included 340 patients (221 boys and 119 girls) examined in the paediatric clinic of the clinical hospital centre osijek and the medical genetics laboratory at the faculty of medicine osijek, who have the diagnosis of id/mr with dysmorphic features and/or multiple malformations. the evaluation included physical examination and collection of family medical history. the physical examination was focused on dysmorphological and neurological evaluation, congenital malformations, somatometric measurements and behavioural evaluations. in patients with neurological symptoms, such as epilepsy and macroor microcephaly, neuroimaging was performed for evaluation of brain malformations. a written informed consent document was signed by the child’s parent/guardian for cytogenetic testing. cytogenetic methods peripheral venous blood was collected by qualified medical staff and sent to the medical genetics laboratory of the faculty of medicine osijek. blood was sampled once by venepuncture and collected into tubes with anticoagulant na-heparin. once received, the samples in the laboratory were identified by a unique laboratory number. cultivation of peripheral blood cells for the purpose of karyotyping was done using a modified moorhead method from 1960 (8). the chromosomes were analysed using a light and fluorescence microscope (olympus bx61) and a digital camera (diagnostic instruments 0.7x hr070-cmt) coupled with the appropriate software (cytovision, applied imaging), in accordance with european guidelines for constitutional cytogenomic analysis, european journal of human genetics (2019) 27:1–16. for the purpose of the fish analysis, in accordance with the suspected structural chromosomal anomalies, we used different locus-specific probes, whole-chromosome paint probes, armspecific probes, centromere probes and subtelomere probes. results a total of 340 patients with id/mr with dysmorphic features and/or multiple malformations were referred for cytogenic evaluation. the age range of the patients was 018 years. we analysed 221 boys (65%) and 119 girls (35%). out of 340 patients, chromosomal abnormalities were found in 76 patients (24.5%). seemedj 2020, vol 4, no. 1 cytogenetic findings intellectual disabilitydysmorphic features 89 southeastern european medical journal, 2020; 4(1) numerical chromosomal aberrations (aneuploidy) were detected in 64 patients (18.8%). the most common type of autosomal aneuploidy was trisomy 21, which was found in 50 patients (14.7%). sex chromosome aneuploidy was detected in 9 patients (2.6%). the results are listed in table 1. table 1. numerical chromosome aberrations (n = number) syndrome karyotype n down syndrome, common type 47,xx,+21 24 47,xy,+21 24 down syndrome, translocation type 46,xy,+21,rob(14;21)(q10;q10)dn 1 down syndrome, mosaicism and translocation type mos 47,xx,+21,der(21;21)(q10;q10)der(21;21)(p10;p10)[71]/46,xx,der(21;21)( q10;q10)der(21;21)(p21;21)(p10;p10)[29]dn 1 edwards syndrome 47,xx,+18 2 patau syndrome 47,xx,+13 2 klinefelter syndrome 47,xxy 5 turner syndrome 45,x 2 triple x syndrome 47,xxx 2 klinefelter syndrome/down syndrome 48,xxy,+21 1 structural chromosomal aberrations were detected in 22 patients (6.5%). patients’ karyotypes and phenotypes are listed in tables 2.a. and 2.b. seemedj 2020, vol 4, no. 1 cytogenetic findings intellectual disabilitydysmorphic features 90 southeastern european medical journal, 2020; 4(1) table 2.a. structural chromosome aberrations in patients with id/mr (m – male; f – female) patient sex phenotype karyotype 1 f large neurocranium, low posterior hairline, broad nasal bridge, low-set ears, synophrys, strabismus, hypertelorism, epicanthal folds, micrognathia, irregular teeth growth, short fingers with clinodactyly. hyperactivity 46,xx,der(2),t(2;4)(p25.1;q31.3)pat 2 m exophthalmos, epicanthal folds, wide nasal bridge, higharched palate, irregular teeth growth, low posterior hairline, low-set ears, long fingers and wide thumbs 46,xy,der(1)t(10;11;1)(10pter→10p11.2::11q25→11q23::1p34.3→1qter)mat ,der(11)t(1;11)(p34.3;q23)mat,t(18;19)(q23;p13.3)dn 3 f microcephaly, short stature, brachycephalic head, microphthalmia, high-arched palate, hypotonia, epilepsy 47,xx,+der(22),t(x;22)(q28;q11.2)mat 4 m microcephaly, antimongoloid slant of eyes, microphthalmia, narrow palpebral fissures, bulbous tip of nose, small mouth and cheilognathopalatoschisis, low-set ears, micropenis, hypospadias, small hands, camptodactyly of the third and fourth finger on both hands, syndactyly of the third and fourth toe and hypoplastic second toe on the right foot. ductus arteriosus 46,xy,del(2)(q31q33) 5 m flat occiput, hypertelorism. tracheoesophageal fistula 46,xy,r(22)dn 6 m sharp hair, low posterior hairline, wide nasal bridge, low-set ears, clinodactyly, hirsutism. atrial septal defect and cryptorchidism 46,xy,der(8)t(4;8)(4pter→4p16.1::8p23.1→8qter)dn seemedj 2020, vol 4, no. 1 cytogenetic findings intellectual disabilitydysmorphic features 91 southeastern european medical journal, 2020; 4(1) table 2.b. microdeletion syndromes (n = number; p = percentage) syndrome karyotype fish n p digeorge syndrome 46,xx 46,xx.ish del(22)(q11.2q11.2)(hira-) 1 1.1% 46,xy 46,xy.ish del(22)(q11.2q11.2)(hira-) 3 prader-willi syndrome 46,xx 46,xx.ish del(15)(q11.2q11.2)(snrpn-) 2 1.5% 46,xy 46,xy.ish del(15)(q11.2q11.2)(snrpn-) 2 prader-willi syndrome – uniparental disomy (upd) 46,xy / 1 williams-beuren syndrome 46,xx 46,xx.ish del (7)(q11.23q11.23)(eln-) 1 0.5% 46,xy 46,xy.ish del (7)(q11.23q11.23)(eln-) 1 cri du chat syndrome 46,xy,del(5)(p15.1)dn 46,xy,del(5)(p15.1).ish del(5)(p15.2)(d5s23,d5s721-)dn 1 0.5% 46,xy,del(5)(p14.2)dn 46,xy,del(5)(p14.2).ish del(5)(p15.2)(d5s23,d5s721-) 1 wolf-hirschhorn syndrome 46,xy,del(4)(p15.3)dn 46,xy,del(4)(p15.3).ish del(4)(p16.3)(whscr)dn 1 0.5% 46,xx,del(4)(p15.32)dn 46,xx,del(4)(p15.32).ish del(4)(p16.3p16.3)( d4s96-, d4z1+, d4s3360-) 1 patient 1’s karyotype is 46,xx,der(2),t(2;4)(p25.1;q31.3)pat. the fish method (probes tel2p, cep2, whsc1, tel4q) shows partial monosomy 2p and partial trisomy 4q. the father’s karyotype is 46,xy,t(2;4)(p25.1;q31.3). the fish method (probes tel2p, cep2, whsc1, tel4q) shows that the father has a balanced reciprocal translocation between chromosomes 2 and 4. the mother has a normal karyotype. patient 2’s karyotype is 46,xy,der(1)t(10;11;1)(10pter→10p11.2::11q25→11q 23::1p34.3→1qter)mat,der(11)t(1;11)(p34.3;q23)mat, t(18;19)(q23;p13.3)dn. the fish method (probes for centromere – 11, 18, subtelomere – 11qter, 18qter, 19pter, wcp – 1, 10, 11) shows an unbalanced complex chromosomal rearrangement that involves chromosomes 1, 10, 11, 18 and 19. derivative chromosome 1 in the patient is inherited from the mother. reciprocal translocation 18;19 is of de novo origin. this is an unbalanced karyotype with 11q25→qter deletion and 10pter→10p11.2 dupllication. the mother’s karyotype is 46,xx,der(1)t(10;11;1)(10pter→10p11.2::11q25→11q 23::1p34.3→1qter),der(10)t(10;11)(p11.2;q25),der(11) t(1;11)(p34.3;q23),t(13;18)(q14;p11.32)dn. the fish method (probes for centromere – 1, 10, 11, 18, subtelomere – 10pter, 11qter, 18pter, 18qter, mcb for chromosome 1, 10, 11, 13, 18, 19, asp – 1p, 11q, 13q, 10p, wcp 13) shows a balanced complex chromosomal rearrangement that involves chromosomes 1, 10, 11, 13 and 18. the karyotypes of the mother’s parents are normal. the sister’s karyotype is 46,xx,t(1;11)(p34.3;q23)?mat,t(13;18)(q14;11.32)mat. the fish method (probes for centromere 10, subtelomere – 10pter, 18pter, 18qter, mcb for chromosome 1 and 11, wcp – 1, 10, 11, 13, 18) shows a balanced complex chromosomal seemedj 2020, vol 4, no. 1 stigmatization of patients with viral infections and mass psychogenic illness 92 southeastern european medical journal, 2020; 4(1) rearrangement that involves chromosomes 1, 11, 13 and 18, but not chromosome 10. the karyotype shows two reciprocal translocations 1;11 and 13;18. since derivative chromosome 1 in the mother includes chromosomes 10;11;1, and in the sister only chromosomes 1;11, it is possible that mother has gonadal mosaicism. the mother and sister have normal phenotypes. patient 3’s karyotype 47,xx,+der(22),t(x;22)(q28;q11.2)mat shows an extra chromosome. the fish method (probes lsi n25 – 22q11.2, lsi shank3 – 22q13.3, cep x dxz1, xqter ms607) shows a derivative chromosome that contains a region from chromosome 22 (22pter→22q11.2) and chromosome x (xq28→xqter). the mother’s karyotype is 46,xx,t(x;22)(q28;q11.2)mat. she has a balanced reciprocal translocation between chromosomes x and 22, which she inherited from her mother, whose karyotype is 46,xx,t(x;22)(q28;q11.2). patient 4’s karyotype is 46,xy,del(2)(q31q33). the mother has a normal karyotype, but we do not know the father’s karyotype, so we cannot detect the origin of the chromosomal aberration. patient 5’s karyotype is 46,xy,r(22)dn. the fish method (chromosome region 22q11.2, 22q12, 22qter) shows deletion of the 22q13 region and de novo formation of ring chromosome 22. patient 6’s karyotype 46,xy,der(8)t(4;8)(4pter→4p16.1::8p23.1→8qter)d n shows an unbalanced translocation between the short arms of chromosomes 4 and 8. the fish method (probes whsc1, cep4, cep8) shows segmental trisomy of the chromosomal segment 4pter-p16.1, and deletion of the chromosomal segment 8pter-p23.1. the parents have normal karyotypes. discussion numerical chromosomal aberrations are common findings that are well-defined clinically, and their prevalence is similar as in the literature. coco and penchaszadeh (9) reported on a cytogenetic study conducted on 200 children with id/mr in argentina. they found chromosomal aberrations in 21% of patients. nasiri et al. (10) reported 23.6% of chromosomal aberrations in their study, similar as in ours. down syndrome is the most common autosomal aneuploidy in our study (14.7%). our results were consistent with many previous studies (11,12). we found double trisomy in one patient (48,xxy,+21). this is a very rare trisomy with the phenotype of down syndrome, as features characteristic of the klinefelter syndrome are not apparent until the post-pubertal stage (13,14,15). there were 0.5% of female patients with the triple x syndrome. this is a very rare syndrome; the phenotype of such women is normal, they are fertile and they have mostly intellectual problems that vary from mild intellectual disabilities, disorders in language development and problems in forming stable interpersonal relationships to severe psychiatric disorders (16). structural chromosomal aberrations seen in our patients are variable and were found in 6.5% of patients. the study conducted by celep et al. (17) reported structural chromosomal aberrations in 4.81% of patients with id/mr and/or multiple congenital malformations. we compared the clinical findings in our patients with similar cases published in the literature. patient 1, with partial monosomy 2p and partial trisomy 4q, has facial dysmorphia and moderate id/mr, but no urogenital and gastrointestinal anomalies or hand anomalies like the patient described in the literature. clinical phenotypes of 2p;4q are variable because the involved breakpoints vary on a case-by-case basis (18). in patient 2, 11q25 deletion is related to developmental delay and facial dysmorphia (exophthalmos, epicanthal folds, wide nasal bridge, high-arched palate, irregular teeth growth, low posterior hairline, low-set ears, long fingers and wide thumbs) (19,20). 10p11.2 dupllication is connected with autistic features (21), which are also present. we presume that novel reciprocal translocation 18;19 does not have an impact on our patient’s phenotype, since it is related to changes caused by acute lymphoblastic leukaemia (22). in patient 3, partial monosomy xq28 could be related to the phenotype listed in table 2.a., seemedj 2020, vol 4, no. 1 stigmatization of patients with viral infections and mass psychogenic illness 93 southeastern european medical journal, 2020; 4(1) since this region contains the mecp2 gene, which causes the rett syndrome, severe epilepsy and psychomotor delay (23). partial trisomy 22q11.23, which is distal from the digeorge syndrome region of the long arm of chromosome 22, could be responsible for other features: growth delay, hypotonia, severe psychomotor delay (24,25). the phenotype of patient 4 (microcephaly, antimongoloid slant of the eyes, microphthalmia, narrow palpebral fissures, bulbous tip of nose, small mouth and cheilognathopalatoschisis, low-set ears, micropenis, hypospadias, small hands, camptodactyly of the third and fourth finger on both hands, syndactyly of the third and fourth toe and hypoplastic second toe on the right foot) is similar to the only published case with del(2)(q31q33) (26). karyotype 46,xy,r(22)dn in patient 6 is known as the phelan-mcdermid syndrome, which includes hypotonia, developmental delay, dysmorphic features (long narrow head, pointed chin, ptosis, deep-set eyes, abnormalities of toes and nails) (27,28). our patient has mild dysmorphia, psychomotor delay and tracheoesophageal fistula which has been successfully corrected, but has no abnormalities of toes and nails or other dysmorphic features typical for the syndrome. the clinical picture could be incomplete, so it would be recommended to perform acgh. we have compared the similarity of patient 7’s features to other patients carrying only a duplication of the distal part of 4p or a deletion of the distal part of 8p or similar, which include low posterior hairline, hirsutism, wide nasal bridge, low-set ears, clinodactyly, atrial septal defect and cryptorchidism (29,30); similar features can be found in our patient. other structural aberrations listed in table 2.b. have a well-defined phenotype (digeorge syndrome, prader-willi syndrome (pws), cri du chat syndrome, wolf-hirschhorn syndrome, angelman syndrome, williams-beuren syndrome). in our study, the most common syndromes are pws (1.5%) and digeorge syndrome (1.1%). digeorge syndrome occurs in 1 in 4.000 people (31) and pws in 1 in 10.000 to 30.000 people (32). it is interesting that we have a very similar percentage of patients with pws and digeorge syndrome, since the prevalence of digeorge syndrome is higher than the prevalence of pws in the general population (31,32). on the other hand, the small percentage of patients with digeorge syndrome in our study could be explained by its variable features. the condition may not be identified in people with mild signs and symptoms, or it may be mistaken for other disorders with overlapping features. conclusion the results of our study show the prevalence of chromosomal aberrations in 24.5% of patients with id/mr and dysmorphia, which confirms similar findings in other screened groups of numerical anomalies. the frequency of aberrations in patients with id/mr in our study was 18.8%, and the most common aberration was ds, as seen in other studies. cytogenetic findings of structural aberrations were 6.5%, which is also similar to other studies. the results of our study show that cytogenetic analysis in patients with id/mr should nowadays be reserved for suspected aneuploidies, since firstline genetic testing for patients with id/mr and especially non-syndromic patients is acgh (33,34). abbreviations mr – mental retardation; 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6(1):042–50. seemedj 2019, vol 3, no. 2 turnover intention among nurses 33 southeastern european medical journal, 2019; 3(2) original article influence of work motivation, work environment and job satisfaction on turnover intention of croatian nurses: a qualitative study 1 eva smokrović 1, 2, ivana gusar 1, 3, dijana hnatešen 1, 4, kristina bačkov 1, 5, antun bajan 1, 6, zvjezdana grozdanović 1, 7, harolt placento 1, 7, boštjan žvanut 8 1 faculty of medicine, josip juraj strossmayer university of osijek, osijek, croatia 2 institution for home healthcare and rehabilitation helena smokrović, rijeka, croatia 3 department of health studies, university of zadar, zadar, croatia 4 clinical hospital center osijek, osijek, croatia 5 general hospital zadar, zadar, croatia 6 department of emergency medicine osijek, osijek, croatia 7 general hospital našice, našice, croatia 8 university of primorska, faculty of health sciences, koper, slovenia corresponding author: ivana gusar, igusar@unizd.hr received: sep 3, 2019; revised version accepted: dec 5, 2019; published: dec 16, 2019 keywords: job satisfaction, motivation, turnover, qualitative research, nurses abstract aim: the purpose of this study was to examine, identify and describe the factors that influence the turnover intention of nurses in croatia. methods: a qualitative descriptive phenomenological study was conducted in june 2018. the participants were 20 registered nurses working full time who were employed in different cities in the republic of croatia, working in different healthcare institutions and in different medical fields. the data were obtained from interviews and were analysed and interpreted using the content analysis method. results: during data analysis, four main ideas emerged as follows: job satisfaction, work motivation, psychological factors (individual) and structural factors – work environment. job satisfaction has been identified as a key factor with direct impact in nurses' turnover intention. work motivation, psychological factors (individual) and structural factors – work environment does not have a direct influence on the nurses' intention to leave their job, but they have a significant indirect impact through job satisfaction. conclusion: recognizing nursing challenges in the healthcare system and the factors which influence the intention of nurses to leave their workplace can help with the development of a clear strategy and retention measures based on the factors that influence the nurses’ turnover intention. (smokrović e, gusar i, hnatešen d, bačkov k, bajan a, grozdanović z, placento h, žvanut b. influence of work motivation, work environment and job satisfaction on turnover intention of croatian nurses: a qualitative study. seemedj 2019; 3(2); 33-44) seemedj 2019, vol 3, no. 2 turnover intention among nurses 34 southeastern european medical journal, 2019; 3(2) introduction the last decade has been marked by a steady increase in the proportion of the elderly, which has in turn increased the need for healthcare (1). the increasing need for healthcare is accompanied by globalization, which, in addition to increased information mobility, also enables greater mobility of people (2). the effects of employees leaving their workplace have caught the attention of many professionals who consider it to be one of the most expensive and difficult challenges faced by modern organizations (3). international migration of nurses in recent years has become a major issue in international political circles (4). croatia’s accession to the european union in 2013 opened a way to other european countries for many croatian professionals, including healthcare professionals. like other european countries, the republic of croatia is currently facing a shortage of nurses and nurse technicians, which has attracted the attention of the croatian media for several years (5-7). another phenomenon that is covertly present, in addition to the lack of professionals with qualifications, is that a great number of nurses do not want to work in the current working conditions. the studies conducted so far indicate a lack of empirical knowledge about the predictors of the intention to leave the workplace. this paper focuses on the influence of certain factors on the intention of nurses to leave their workplace, institution, or country. the phenomenon of abandonment of the nursing profession, resulting in a decline in the number of educated nurses, poses a significant problem for the functioning of healthcare systems in many countries (2, 8), and especially in the less developed countries of the european union (eu) (9, 10). the financial crisis, limitations of the national budget, reforms of the healthcare system, the workload of healthcare professionals, the decrease (or stagnation) of personal incomes and unpaid overtime represent just some of the factors that have affected the nursing profession both all over the world and in croatia (2). the results of numerous studies conducted on the topic indicate that a growing number of nurses are leaving their workplace (11, 12), going to a different institution, or even leaving the nursing profession altogether. employee’s intention to leave the primary workplace is defined as the deliberate, voluntary and selfinitiated change of job or institution. the intention to leave actually reflects an employee’s attitude toward their current workplace or organization (13). from the employee’s perspective, the intention to leave can be seen as a positive phenomenon. reasons for positive perception of the above relate to better job offers that include higher income and/or intangible benefits, such as greater autonomy and new challenges (14). moyce, lash and de leon siantz (2016) cite the so called “push and pull” factors as reasons why nurses leave their workplace. “push” factors are the factors that encourage nurses to leave and “pull” factors are the factors that attract them to new opportunities and challenges (2). among the most common factors that drive nurses to leave are dissatisfaction with the working conditions, stress and emotional exhaustion, while the factors that attract them are higher income, greater autonomy, recognition and respect within a team of healthcare professionals (2). in a study conducted in singapore on a sample of 814 nurses, goh and lopez (2016) describe leadership skills and work environment as the most important predictors of the nurses’ intention to leave (15). an individual’s statement of the intention to leave has, in previous research, been proved to be a significant predictor of their leaving the workplace (16). simply put, an individual who verbally expresses their intention to leave a job is significantly more likely to leave the job than others. the validity of the above predictor has been highlighted in several studies of nurses’ intention to leave (17– 19). brewer and kovner (2014) propose and describe the concept of nurse turnover based on seemedj 2019, vol 3, no. 2 turnover intention among nurses 35 southeastern european medical journal, 2019; 3(2) a model that includes job satisfaction, organizational commitment, intention to leave and intention to seek a new position. official data on croatian nurses who have left the workplace or migrated are not available; however, according to kovačević barišić and lepan stefančić (2015), around 400-500 nurses left croatia shortly after croatia joined the eu, in 2014 and 2015. identifying and understanding the factors that significantly affect job satisfaction and the level of nurses’ work motivation is useful for preventing dissatisfaction and decreasing the intention of nurses to leave their current workplace or profession (20). barać et al. (2015) performed a study to that effect on croatian nurses, but it focused on job satisfaction, which is a predictor of nurses leaving the workplace (21). smokrović et al. (2019) conducted a quantitative study, where the impacts of work motivation, organisation, and absenteeism on job satisfaction and intention to leave were considered (22). however, the aforementioned studies assessed the impact of predefined factors identified in a review of the literature on job satisfaction and turnover intention. the participants did not have the option of adding extra factors. hence, the goal of this study was to identify, through the use of the qualitative research method, potential factors that have an impact on job satisfaction and turnover by considering the opinions of croatian nurses. we posed the following research question: which factors influence the turnover intention of croatian nurses? methods a descriptive qualitative study was conducted in accordance with the guidelines on qualitative research (23). study design a qualitative approach was applied in order for us to gain a profound understanding of the nurses’ opinions and attitudes in the context of work motivation, work environment, job satisfaction and intention to leave. after a thorough review of the available literature and clearly defined ideas, certain claims/situations were established based on the existing theories and insights gained into each particular construct. the central phenomenon which was examined was the experience of nurses related to work motivation, job satisfaction and work environment, and the importance and impact of these concepts on turnover intention were examined as well. participants in order to obtain a heterogeneous sample of participants, the study was conducted in different croatian cities: rijeka (five participants), osijek (five participants), zadar (five participants), and našice (five participants); in different healthcare institutions (primary – five participants, secondary – 10 participants, and tertiary – five participants); 15 participants were employed in public, and five in private healthcare institutions); and in different nursing fields: 15 were working in healthcare centres, four were employed in departments of internal medicine, four were surgical nurses, two were employed in gynaecology departments, two were from paediatrics, one was employed in the department of neurology, one was employed in the department of oncology and one was working in the department of urology. nurses were randomly selected from the list of nurses employed in each of the participating healthcare institutions. the list of nurses was formed in accordance with the following predefined inclusion criteria: 1) a registered nurse with a valid license, 2) a minimum of five years of work experience in the profession in the institution of current employment, 4) full-time employment, 5) open-ended employment contract, 6) voluntary participation in the study. a total of 20 nurses were selected who met the above criteria and who expressed their consent to participate in the study. data collection seemedj 2019, vol 3, no. 2 turnover intention among nurses 36 southeastern european medical journal, 2019; 3(2) data were collected using a semi-structured face-to-face interview conducted between 4 june 2018 and 22 june 2019. interviews were conducted by seven experts in the field of clinical and medical sciences, nursing sciences in collaboration with psychology and information science experts. participants were interviewed in a room outside of their usual work environment, without external interference and without the presence of others. just before conducting the interview, the researchers were reminded of the importance of using comprehensible language in communication, interactivity, changing the order of questions/statements or asking additional questions to get to the essence of the topic, and of the obligation to verify that they understood their interviewees’ statements. prior to each interview, the purpose of the study and the reasons for conducting it were explained to the participants. the participants’ anonymity was guaranteed. participants voluntarily agreed to participate either verbally or in writing. likewise, with prior permission given by the participants, for the purpose of clearer understanding and more detailed processing of data, the interviews were recorded and transcribed verbatim. during the interview, each researcher presented a total of nine possible (fictional) situations with fictional characters – nurses. the fictional situations were designed by considering everyday real-life/work situations. after the individual statement/situation was read and presented to the participants, they had the opportunity to reflect on their position and think of a possible solution to the situation and then verbalize their opinion. after the presentation of the situations, it was left to the participants to seek further clarification regarding the presented situation. after each participant responded to the situation, a mandatory additional question was asked: do you think the reasons would be different for male nurses? the interviews lasted between 30 and 60 minutes and there was no need to repeat any of them. data analysis data saturation was achieved in the 11th interview. four additional interviews were processed to increase the quality of the study, and in total 15 interviews were analysed. all interviews were carefully transcribed and analysed, where the researchers analysed each sentence in detail according to van manen’s (2002) detailed analysis approach (24). colaizzi’s (1978) seven-step approach was also used to ensure an in depth analysis of interview data using a clear and systematic approach (25). each interview was separately coded by at least two experts. in the first step, a naive reading of interview transcripts was performed, followed by axial coding, where codes were refined, and selective coding, where the final coding was performed. after revising each transcript, the researchers consulted one another and performed the final categorization, i.e. they repeated selective coding. the researchers extracted illustrative quotes, which were classified into separate categories. for example:“… she is primarily satisfied because she loves her job, loves helping people who need help: it makes her feel happy, satisfied, fulfilled because helping someone who needs it is of great importance for her personal satisfaction. interpersonal relationships and the atmosphere in the department where she works are good, as are the overall working conditions …” (participant 5, female, 7 years of service). individual ideas were identified and a detailed description of each idea was then drawn up. to verify the accuracy of interpretation, interview transcripts were read three times. in the final step, there was no need to include any further changes based on feedback from the research participants. data saturation was achieved in the 11th interview. four additional interviews were processed to increase the quality of the study, and in total 15 interviews were analysed. all interviews were carefully transcribed and analysed, where the researchers analysed each sentence in detail according to van manen’s (2002) detailed analysis approach (24). seemedj 2019, vol 3, no. 2 turnover intention among nurses 37 southeastern european medical journal, 2019; 3(2) colaizzi’s (1978) seven-step approach was also used to ensure an in depth analysis of interview data using a clear and systematic approach (25). each interview was separately coded by at least two experts. in the first step, a naive reading of interview transcripts was performed, followed by axial coding, where codes were refined, and selective coding, where the final coding was performed. after revising each transcript, the researchers consulted one another and performed the final categorization, i.e. they repeated selective coding. the researchers extracted illustrative quotes, which were classified into separate categories. ethical aspects all participants, after being properly informed of the purpose of the research, voluntarily agreed to participate in the research. all of them signed a consent form for participating in the survey or expressed their consent orally. the interviewee’s name was not mentioned at any time during the interview to protect the privacy of the participants. all participants were made aware of the fact that they had the right and the option to refuse to give an answer to or opinion on a particular statement/situation before the start of the interview and they were aware of the fact that they could stop the interview at any time. results demographic data all participants included in this study were female, employed full time and were registered nurses. the youngest participant was 27 and the oldest was 48 years old. the average age of the participants was 33, with 6.5 to 28 years of work experience. they all worked and lived in croatia and were native speakers of croatian. thematic analysis and categorization thematic analysis of the content by code group resulted in a division into four categories. each category contained a number of subcategories, all of which have a significant impact on the main idea (table 1). participants’ opinions for each category were further expanded and compared with other studies conducted in europe and the rest of the world that have been published and made available so far, which will be presented in the discussion section. table 1. nurses’ intention to leave – thematic content analysis by category and subcategory category subcategory job satisfaction positive attitudes * autonomy * acknowledgment of a job well done * stress ** possibility of advancement * wrong choice of profession ** work motivation identification with the job * intrinsic motivation * extrinsic motivation (material) * psychological factors (individual) family factors * younger age * change of residence * family migration * structural factors – work environment working conditions ** availability of materials, sufficient time and employees ** conflict in the workplace ** managers’ ability to manage and support * *positive effect; **negative effect seemedj 2019, vol 3, no. 2 turnover intention among nurses 38 southeastern european medical journal, 2019; 3(2) presentation of participants’ responses job satisfaction was most influenced by: good interpersonal relationships, complexity of the work, team atmosphere and positive team attitudes (n = 13); doing the job you love (n = 9); good organization and working conditions, job security, regular income (n = 7); and identifying with nursing as a profession because it makes me happy, satisfied and fulfilled (n = 5). the participants cite positive internal motivation, capabilities and accessibility of their superiors as other reasons. thirteen participants believe that there is no difference in the factors of job satisfaction between men and women; however, two participants state that by acquiring the skills, a male nurse changes jobs more quickly and easily and their opinions are more respected than the opinions of female nurse. job dissatisfaction was most influenced by: dissatisfaction with the work environment, organization of work, night shifts, performing tasks outside the scope of the job, low salaries, non-recognition of professional qualifications, conflicts in the work environment (n = 15); personal participation in internal conflicts in the work environment (n = 12); privileged positions of superiors and inequality (n = 4); choosing the wrong profession (n = 2). other reasons indicated were that the superior is not a good manager and that the nursing profession is not appreciated enough. the majority of participants (n = 10) feels that there is no difference in job dissatisfaction factors between males and females; however, some participants state that men would switch jobs if they were not satisfied, especially if better pay was involved. change of workplace within an institution is most often due to: poor interpersonal relationships and communication in the department (n = 9); advancement (n = 5); health problems (n = 5); being tired of one’s job (n = 3); compromised position and protection (n = 2). the opinion of 11 participants is that there are no differences of opinion between men and women, but other participants state that men have to face new challenges in their desire for promotion and a better workplace. nurses decide to change the institution where they work because of: better working conditions and interpersonal relationships (n = 9); proximity to work (e.g. relocation) and therefore more family time (n = 9); financial situation, as well as non-recognition of a higher education degree (n = 6). the majority of participants (n = 10) believe that there is no difference in reasoning between male and female nurses; however, some say that male nurses would be looking for new challenges and better pay. leaving the nursing profession is most often due to: wrong choice of profession, poor working conditions, interpersonal relationships and financial situation (n = 9); stress (n = 3). eleven participants believe that there are no differences of opinion between males and females, while other participants note financial problems as a reason for changing their profession. nurses do not intend to leave their department, institution or profession because they are: satisfied with their job, working conditions, interpersonal relationships and superiors (n = 14); in addition to the above assertion, five participants state that they have a fear of a new environment. two participants cite status as a reason. fifteen participants feel that there are no differences between the opinions of male and female persons. nurses decide to go abroad for: financial reasons (n = 12); better working conditions and healthcare system (n = 11). as for other reasons, they state that younger people are more likely to choose to leave, as are those who have already family abroad, and that the profession is more appreciated abroad. seemedj 2019, vol 3, no. 2 turnover intention among nurses 39 southeastern european medical journal, 2019; 3(2) direct effect indirect effect thirteen participants feel that there are no differences between male and female persons; however, two participants state that it is easier for men to leave. the most common reasons for nurses only doing their job enough to “satisfy the form” are: there are no rewards or praise, which is demotivating (n = 12); routine and saturation (n = 6). participants also indicate selection of the wrong profession and early retirement of employees. a total of fifteen participants believe that there are no differences between the opinions of male and female persons. nurses are pleased with their superiors because of: systematization and regulated scope of work and good organization in the department; leadership, good communication skills, understanding of team needs and teamwork, managing the department professionally and responsibly (n = 15). thirteen participants are of the opinion that there are no differences between male and female persons; however, two participants note that more tolerance is shown for male than female nurses. presentation of the main category relationships on nurses’ intention to leave by further analysing the results obtained regarding the defined main categories and subcategories, we detected categories that have a direct impact on nurses’ intention to leave. additionally, we also present the categories that directly affect job satisfaction and indirectly affect the nurses’ intention of leaving. the results are shown in figure 1.. the ability of a manager, support of superiors, interpersonal relations and working conditions structural factors (work environment) work motivation job satisfaction turnover intention individual psychological factors figure 1. main category and subcategory influences on nurses’ turnover intention discussion the results of the study indicate that four main categories of factors are important for nurses’ intention of quitting their jobs in croatia. the first category is job satisfaction, which has been identified as a key factor in nurses’ turnover intention, as has been confirmed in a number of previous studies conducted in croatia and seemedj 2019, vol 3, no. 2 turnover intention among nurses 40 southeastern european medical journal, 2019; 3(2) worldwide (3, 16, 22, 26). the results indicate that dissatisfaction with work and the work environment is a clear indication that nurses will leave their jobs in the future. a survey conducted in turkey in 2015 recognizes job satisfaction as an important factor and states that 61% of nurses intending to leave their job register a moderate level of satisfaction (12). positive attitudes, autonomy, recognition of a job well done, and the ability to advance in the workplace were highlighted as subcategories of job satisfaction, which has been confirmed in previous research. in a recent croatian survey, barać et al. (2017) also describe nurses’ job satisfaction and cite that nurses who are more committed to their work have greater job satisfaction (27). the level of job satisfaction is closely related to work motivation (27, 28), which represents the second category in our research. work motivation does not have a direct influence on the nurses’ intention to leave their job, but it has a significant indirect impact on job satisfaction. similar results were previously reported in a study conducted in croatia (22). singh and tiwari also found a correlation between job satisfaction and work motivation in their research. they claim that increasing job satisfaction increases work motivation and vice versa. employee satisfaction thus increases work motivation, while dissatisfaction effects motivation decline (27). the results indicate a significant impact of work motivation through the subcategories of identification, intrinsic motivation, and extrinsic (material) goals. gagné et al. (2015) state that an autonomous type of motivation, especially intrinsic, which is also the preferred type of motivation, is related to job satisfaction and the intention to leave (29). the same results were also found in a study conducted in the netherlands (30), which aimed to examine the connection between work stressors and the reactions of employed nurses. in another study, singh and tiwari (2007) describe that lack of job satisfaction is a predictor of leaving, driven by an individual’s motivation (27). goh and lopez (2016) especially emphasize the impact of intrinsic and extrinsic factors on employee satisfaction, which is also highlighted in our results in the category of work motivation (15). in contrast with the results of smokrović et al. (2019), where nurses who identified with the profession felt lower job satisfaction, our results indicate a positive relationship between identifying with the profession, intrinsic and extrinsic motivation and work motivation. since extrinsic motivation plays a significant role in overall satisfaction, the impact of a system of support, rewards and bonuses on employee motivation should not be neglected. if the nurse is acknowledged and supported by the staff and superiors, their self-esteem and satisfaction at work will grow. this reduces the possibility that the nurse will consider leaving the workplace. in a study conducted in jordan, mahmoud alhussami (2008) also cites a strong relationship between job satisfaction and support from superiors (31). the same results were found in a study conducted in croatia, where the authors said that managers’ support is a valid predictor of job satisfaction (22). the majority of participants emphasize extrinsic material motivation and inadequate material income in their responses as a reason for dissatisfaction and demotivation. they feel that material motivation has a significant impact on the intention to leave the country and the workplace. in previous research, material welfare was also recognized as a significant factor (8). job satisfaction is also closely related to employees’ personal characteristics (29, 32) and the work environment (28, 29, 33, 34). our results show that individual psychological factors of employees, work environment and work motivation are important for achieving the level of ultimate satisfaction. in their literature review, lu, while and louise barriball (2005) highlight working conditions, working relationships, salary, promotion opportunities, job security, accountability, recognition by superiors and overtime as the most common factors that have a direct impact on satisfaction. additionally, toode, routasalo and suominen (2011) state that personal characteristics and priorities, in addition to the internal seemedj 2019, vol 3, no. 2 turnover intention among nurses 41 southeastern european medical journal, 2019; 3(2) psychological state, influence work motivation. the results of this study reveal that personal characteristics of employees are extremely important predictors of their intention to leave. the study implies that the intention to leave is not necessarily related to the institution of employment, work organization, collective or motivation, and we believe that personal characteristics need to be singled out and emphasized as a separate factor in the intention to leave. in this study, we categorized individual psychological factors that have a significant indirect impact on the intention to leave, such as family, age, change of residence, and family migration; this was also partially confirmed in a previous study (22). employees report that the choice to leave is easier for younger people, as well as for those whose family members have previously migrated. different results specific for some eu countries have also been published in a study conducted in 10 eu countries that aimed to discover the factors that influence leaving the workplace (1). as the last category which, in addition to indirect influence through job satisfaction, also has a direct impact on nurses’ intention to leave, the work environment was also found to be a factor in our study, and it was also highlighted in studies mentioned above (1, 22, 28, 31, 34). masum et al. (2016) conducted their research in private healthcare facilities in turkey and, in addition to the factors mentioned above, specifically emphasized the importance of the work environment and the relationship with colleagues and superiors. our results also confirm that a strong correlation exists between the characteristics and quality of the work environment and the decision to leave the workplace. the participants stressed material and human resources, e.g. availability of materials, working conditions, sufficient time and number of employees, as important determinants of the work environment. as in previous studies, the possibility of further advancement and qualities of the manager were classified under the category of work environment (1, 34). a chinese study conducted by wang et al. (2015) examined the impact of nurses’ work environment on job satisfaction and considered the work environment to be a key factor in an organization. they found that it has a stronger impact on job satisfaction than any other organizational or personal factor. the same results were obtained in a study conducted in the united states of america (33). as stated by latham, hogan and ringl (2008), work environment is highlighted as a crucial factor in the intention to leave or remain in the workplace, along with job satisfaction (35). employees will find it much more difficult to leave the workplace if they work in a good, positive environment where they do not encounter daily conflict situations. the ability to advance, the superiors’ ability to lead and manage, and support by the superiors are the factors that are most commonly mentioned as factors that influence the decision to leave in this category. in conclusion, the departure of nurses from croatia or the workplace poses a major challenge to the healthcare system. the results of this study are thus aimed at managers who, in the present-day migration of all healthcare professionals, including nurses, should prioritize the development of a clear strategy and measures for retention of nurses based on the factors that influence their intention to leave. decision makers in healthcare must take all steps that are necessary for creating a positive work environment and stimulating employee motivation and satisfaction. this research was conducted on a relatively small sample of nurses, and we believe that in the near future a detailed and extensive combined examination of the reasons for the turnover intention of nurses in the republic of croatia, covering all territorial regions, will be required. the proposed research would certainly provide detailed insight into the above factors, which would lead to a deeper understanding of this phenomenon. seemedj 2019, vol 3, no. 2 turnover intention among nurses 42 southeastern european medical journal, 2019; 3(2) acknowledgement. we would like to thank all the participants in this research who contributed to the development of knowledge in the field of nursing with their answers. we would also like to thank maja frencl žvanut for her support in developing this study. this paper is part of a seminar project of students at the postgraduate doctoral study in biomedicine and health at the faculty of medicine in osijek. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. heinen mm, van achterberg t, schwendimann r, zander b, matthews a, kózka m, et al. nurses’ intention to leave their profession: a cross sectional observational study in 10 european countries. int j nurs stud. 2013;50(2):174–84. 2. moyce s, lash r, de leon siantz m lou. migration experiences of foreign educated nurses: a systematic review of the literature. j transcult nurs. 2016;27(2):181–8. 3. rizwan m. determinants of employees intention to leave. determinants employees intent to leave [internet]. 2014;4(3):1–19. available from: http://www.macrothink.org/journal/index.php /ijhrs/article/viewfile/5871/4760 4. brewer cs, kovner ct. intersection of migration and turnover theories-what can we learn? 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guidelines for researchers, mentors and reviewers. annu soc work. 2017;21(2):263–93. 24. van manen m. writing in the dark: phenomenological studies in interpretative inquiry. ontaio, canada: althouse press; 2002. 25. morrow r, rodriguez a, king n. collaizzi’s descriptive phenomenological method. psychologist. 2015;28(8):643–4. 26. lu h, while ae, louise barriball k. job satisfaction among nurses: a literature review. int j nurs stud. 2005;42(2):211–27. 27. singh sk, tiwari v. relationship between motivation and job satisfaction of the white collar employees: a case study. j educ policy. 2007;2(7):1–10. 28. lorber m, skela savič b. job satisfaction of nurses and identifying factors of job satisfaction in slovenian hospitals. croat med j. 2012;53(3):263–70. 29. gagné m, forest j, vansteenkiste m, crevierl, broeck a van den, aspeli ak, et al. the multidimensional work motivation scale : validation evidence in seven languages and nine countries the multidimensional work motivation scale : validation evidence in. 2015;0643(january 2016). 30. jantschek n, jantschek i, kunzendorf s, blomhoff s, malt uf, jahnsen j. the inspire study : do personality traits predict general quality of life ( short form-36 ) in distressed patients with ulcerative colitis and crohn ’ s disease ? scand j gastroenterol. 2008;(0027):1505–13. 31. mahmoud al-hussami. a study of nurses’ job satisfaction: the relationship to organizational commitment, perceived organizational support, transactional leadership, transformational leadership, and seemedj 2020, vol 4, no. 1 stigmatization of patients with viral infections and mass psychogenic illness 44 southeastern european medical journal, 2020; 4(1) level of education. eur j sci res. 2008;22(2):286– 95. 32. dywili m. the relationship between occupational stress and intentions to quit among employees at nkonkobe municipality, south africa. bus econ j [internet]. 2015;06(02). available from: https://www.omicsonline.com/openaccess/the-relationship-betweenoccupational-stress-and-intentions-toquitamong-employees-at-nkonkobemunicipality-south-africa-2151-62191000146.php?aid=48714 33. choi j, flynn l, aiken lh. nursing practice environment and registered nurses’job satisfaction in nursing homes. gerontologist. 2012;52(4):484–92. 34. sojane js, klopper hc, coetzee sk. leadership, job satisfaction and intention to leave among registered nurses in the north west and free state provinces of south africa. curationis. 2016;39(1):1–10. 35. latham cl, hogan m, ringl k. nurses supporting nurses. nurs adm q. 2008;32(1):27– 39. seemedj 2021, vol 5, no. 2 blood group and covid-19 in patients with malignant disease 1 southeastern european medical journal, 2021; 5(2) original article blood group and covid-19 transmission and mortality in patients with malignant disease 1 senar ebinç *1, zeynep oruç1, ziya kalkan1, zuhat urakçı1, muhammet ali kaplan1, mehmet küçüköner1, abdurrahman işıkdoğan1 1 department of medical oncology, dicle university, diyarbakır, turkey *corresponding author: senar ebinç, senarebinc@gmail.com received: apr 18, 2021; revised version accepted: oct 10, 2021; published: nov 26, 2021 keywords: covid-19, abo blood group system, neoplasm abstract aim: the study aimed to investigate risk factors affecting the transmission of and mortality from covid-19 in patients diagnosed with a malignant disease. in this context, abo blood groups, gender, age, type of malignant disease, type of anti-tumoral agents, comorbidities and stage were examined. materials and methods: files of 1,256 patients who presented to our clinic between march and december 2020 were retrospectively reviewed. patients diagnosed with a malignant disease who became infected with covid-19 and those who did not were compared with regard to demographic, clinical characteristics and laboratory results (status of having been infected with covid-19, abo blood groups). results: of 1256 patients in total, 72 (5.7%) were diagnosed with covid-19. median age of cancer patients infected with covid-19 was 53 years (18-80). the most common types of cancer included gastrointestinal cancer (22.2%), breast cancer (20.8%), genitourinary cancer (20.8%) and lung cancer (16.7%). of the patients diagnosed with covid-19, 18.1% (n=13) died. multivariate analysis identified disease stage as an independent prognostic factor for the risk of mortality [hr: 0.07, 95% ci (0.0070.74), (p=0.02)]. a comparison of patients who became infected with covid-19 and those who did not with regard to abo blood groups (p=0.39) showed no statistically significant differences between the two groups. there was also no correlation between abo blood groups and the risk of covid-19related mortality (p=0.83). conclusion: in patients suffering from malignant diseases, the abo blood type exhibited no correlation with the risk of covid-19 transmission and mortality. this study determined the presence of metastatic disease as a negative prognostic factor. patients suffering from a metastatic malignant disease represent a high risk group for covid-19 and should be treated using all necessary precautions. (ebinç s, oruç z, kalkan z, urakçı z, kaplan ma, küçüköner m, işıkdoğan a. blood group and covid19 transmission and mortality in patients with malignant disease. seemedj 2021; 5(2); 1-8) seemedj 2021, vol 5, no. 2 blood group and covid-19 in patients with malignant disease 2 southeastern european medical journal, 2021; 5(2) introduction sars-cov-2 infection, the first cases of which were reported in the people’s republic of china in late 2019, spread rapidly within a short period of time, marking the beginning of the covid-19 pandemic. during this period, which has impacted almost all populations, patients diagnosed with a malignant disease were included among high-risk patient groups (1,2). besides cancer, conditions such as immunosuppression, presence of comorbidities and advanced age resulted in these patients being included in groups impacted more severely by the infection (3). frequent hospital visits are also among the factors elevating the risk of covid-19 transmission in this population (4). the genetically coded abo blood group system is a phenotypic reflection of a genetic makeup. these antigens may serve as a protective factor against certain diseases and as a predisposing factor for others. furthermore, the abo blood group system was found to be associated with multiple infectious diseases. in some studies, it has been reported that anaemia is more common in a blood group in plasmodium falciparum and that hiv ag/ab is more common in a blood group (5, 6). relationships between many infectious pathogens and blood group antigens and anti-a/b antibodies have been investigated in extensive analyses (7). for example, some human immunodeficiency virus (hiv) and gp120 isolates can react with blood group active monoclonal antibodies and lectins (8). many studies have been conducted to evaluate the relationship between covid-19 infections and the abo blood group system. some studies have found that women with a blood group are more susceptible to covid-19 infections, while much lower covid-19 infection rates were found in those with o blood group (9). in a study conducted during the hong kong epidemic, it was argued that o blood group is more resistant to the transmission of severe acute respiratory syndrome coronavirus (sarscov) and that the risk of transmission is higher in a, b and ab blood groups (10). like hiv, sarscov targets the host cell via adhesion glycoproteins. it has been suggested that sarscov targets the respiratory and gastrointestinal mucosa with its spike (s) protein and that hightitre anti-a monoclonal antibody can block the s protein (11). based on this information, the status of blood groups in the transmission of and mortality from covid-19 was examined in patients suffering from malignant diseases. materials and methods files of patients diagnosed with a malignant disease who presented to our medical oncology clinic between march 2020 and december 2020 were retrospectively reviewed. demographic, clinical characteristics of the patients, data about their treatment and laboratory results (status of having been infected with covid-19, covid-19 pcr test results, abo blood group results) were obtained from the hospital records system. patients in whom the presence of a sars-cov2 infection was confirmed clinically and by laboratory results (positive pcr test) were considered covid-19 cases. patients diagnosed with covid-19 were defined as the case group and patients not diagnosed with covid-19 were defined as the control group. clinical and laboratory characteristics of the groups and their survival outcomes were evaluated. information about the blood group of 1,256 patients who presented to our clinic during the pandemic was available. of these patients, 72 contracted covid-19 and 13 patients in the patient group that contracted covid-19 died. all statistical analyses were performed based on the specified numbers of patients. patients were categorised based on age, gender, presence of comorbid diseases, type of cancer diagnosis, disease stage (metastatic/non-metastatic), treatment agents they received (chemotherapy, immunotherapy, tyrosine kinase inhibitors, antihormonal therapies) and blood groups (abo, rh). the case and control groups were compared according to these parameters. also, the case groups were further categorised as survivors and non-survivors. seemedj 2021, vol 5, no. 2 blood group and covid-19 in patients with malignant disease 3 southeastern european medical journal, 2021; 5(2) mann-whitney u test and logistic regression analysis were used for the analyses of nonnormally distributed or non-parametric variables. a 95% confidence interval and significance value of p<0.05 were used. table 1. mortality risk for patients with covid 19 + according to disease and treatment characteristics n (%) nonsurvivor (n,%) survivor (n,%) p value all patients 72 13 (18.1%) 59 (81.9%) age (median, yrs) 53 (18-80) 46 (18-80) 55 (18-75) 0.23** gender 0.41* male 37 (51.4%) 8 (61.5%) 29 (49.2%) woman 35 (48.6%) 5 (38.5%) 30 (50.8%) comorbiditi es 0.79* yes 30 (41.7%) 5 (38.5%) 25 (42.4%) no 42 (58.3%) 8 (61.5%) 34 (57.6%) diagnosis 0.65* brain 3 (4.2%) 0 (0%) 3 (5.1%) gus 15 (20.8%) 1 (7.7%) 14 (23.7%) gi̇ 16 (22.2%) 5 (38.5%) 11 (18.6%) soft tissue 5 (6.9%) 1 (7.7%) 4 (6.8%) breast 15 (20.8%) 2 (15.4%) 13 (22%) lung/pleura 12 (16.7%) 3 (23.1%) 9 (15.3%) head and neck 2 (2.8%) 0 (0%) 2 (3.4%) others 4 (5.6%) 1 (7.7%) 3 (5.1%) stage 0.01* metastatic 46 (63.9%) 12 (92.3%) 34 (57.6%) nonmetastatic 26 (36.1%) 1 (7.7%) 25 (42.2%) anti tumoral agents (n:61) 0.79* chemotherapy 36 (59%) 5 (50%) 31 (60%) tki̇s 9 (14.8%) 2 (20%) 7 (13%) i̇mmunotherapy 3 (4.9%) 1 (10%) 2 (3.9%) hormonal therapy 13 (21.3%) 2 (20%) 11 (21.6%) tki̇: tyrosine kinase inhibitors, gus: genitourinary system, gi̇: gastrointestinal truct, * chi-square test, ** independent samples t test. seemedj 2021, vol 5, no. 2 blood group and covid-19 in patients with malignant disease 4 southeastern european medical journal, 2021; 5(2) statistical anaylsis data were statistically analysed using the spss 18.0 package program. descriptive statistics were used to evaluate patient characteristics and the frequencies of the parameters. student’s t-test was used for normally distributed numeric variables. a chi-squared test, fisher’s exact test, results the study included 1,256 patients whose data could be obtained. during the pandemic, 72 (5.7%) patients were diagnosed with a covid-19 infection. general characteristics of the patients are presented in table 1. table 2: covid-19 transmission and mortality status according to blood groups a ll p a ti e n ts c o v id -1 9 + n c o v id (+ ) c o n tr o l x 2 h r (9 5 % c i) p * n n o n su rv iv o r s u rv iv o r x 2 h r (9 5 % c i) p * a ll p a ti e n ts 12 5 6 7 2 (5 .7 % ) 11 8 4 (9 4 .4 % ) 7 2 13 (1 8 % ) 5 9 (8 2% ) b lo o d g ro u p s ( a -b -o ) 0 .3 8 0 .3 9 0 .4 0 0 .8 3 o 4 3 0 (3 4 .2 % ) 28 (3 8 .8 % ) 4 0 2 (3 3 .9 % ) re fe re n c e 28 (3 8 .9 % ) 6 (4 6 .2 % ) 22 (3 7 .3 % ) re fe re n c e a 4 8 8 (3 8 .9 % ) 21 (2 9 .1 % ) 4 6 7 (3 9 .4 % ) 0 .6 4 (0 .3 6 1. 15 ) 0 .1 4 21 (2 9 .2 % ) 3 (2 3 .1 % ) 18 (3 0 .5 % ) 0 .6 1 (0 .1 3 2. 7 9 ) 0 .5 2 b 22 2 (1 7 .7 % ) 15 (2 0 .8 % ) 20 7 (1 7 .4 % ) 1. 0 4 (0 .5 4 1. 9 9 ) 0 .9 0 15 (2 0 .8 % ) 4 (3 0 .7 % ) 11 (1 8 .6 % ) 1. 3 3 (0 .3 15 .7 2) 0 .6 9 a b 11 6 (9 .2 % ) 8 (1 1. 1% ) 10 8 (9 .1 % ) 1. 0 6 (0 .4 7 2. 4 0 ) 0 .8 8 8 (1 1. 1% ) 0 (0 % ) 8 (1 3 .6 % ) 0 .0 0 0 .9 9 r h g ro u p 0 .1 9 0 .6 6 (0 .3 5 1. 23 ) 0 .1 9 0 .7 8 1. 26 (0 .2 4 6 .5 1) 0 .7 8 p o si ti v e 10 9 2 (8 6 .9 % ) 5 9 (8 1. 9 % ) 10 3 3 (8 7 .2 % ) 5 9 (8 1. 9 % ) 11 (8 4 .6 % ) 4 8 (8 1. 4 % ) n e g a ti v e 16 4 (1 3 .1 % ) 13 (1 8 .1 % ) 15 1 (1 2. 8 % ) 13 (1 8 .1 % ) 2 (1 5 .4 % ) 11 (1 8 .6 % ) x2:chi-square test, *binary logistic regression seemedj 2021, vol 5, no. 2 blood group and covid-19 in patients with malignant disease 5 southeastern european medical journal, 2021; 5(2) median age of patients who contracted covid19 was 56 years. male patients comprised 51.4% (n=37) of the cases. the most common types of cancer in covid-19-positive patients included gastrointestinal cancer (22.2%), breast cancer (20.8%), genitourinary cancer (20.8%) and lung cancer (16.7%). univariate and multivariate analyses showed that age, gender, comorbidity status and type of primary diagnosis had no effect on mortality in the patients infected with covid-19. of 61 patients in active treatment, 59% (n=36) received chemotherapy, 21% (n=13) received hormonal therapies, 14.8% (n=9) received tyrosine kinase inhibitor and 4.9% (n=3) received immunotherapy agents. a comparison of patients with regard to treatment they received showed no statistically significant differences between treatment agents in terms of mortality rates (p=0.79). of 1,256 patients evaluated with regard to abo and rh blood groups, 72 patients diagnosed with covid-19 were defined as the case group and 1,184 covid-19-negative patients were defined as the control group. in the case group, the rates of o, a, b and ab blood groups were 38.8% (n=28), 29.1% (n=21), 20.8% (n=15) and 11.1% (n=8), respectively. regarding the case group, 81.9% of the patients (n=59) were rh-positive and 18.1% (n=13) were rh-negative. meanwhile, in the control group, the rates of o, a, b and ab blood groups were 33.9% (n=402), 39.4% (n=467), 17.4% (n=207) and 9.1% (n=108), respectively. in the control group, 87.2% (n=1,033) of patients were rh-positive and 12.8% (n=151) were rh-negative. there was no statistically significant difference arising from abo and rh groups between the case and control groups with regard to the risk of covid-19 transmission or among covid-19positive patients with regard to the risk of mortality. details regarding blood groups are specified in table 2. the evaluation of covid-19-positive patients in multivariate analyses according to the stage of disease showed that patients had a higher mortality risk in the metastatic stage compared with the non-metastatic stage [mortality rate of 26% in the metastatic stage, 3.8% in the nonmetastatic stage (hr: 0.07, 95% ci (0.007-0.74), (p=0.02)]. results of multivariate analysis are presented in table 3. table 3. multivariate analysis for mortality risk in patients with covid 19 (+) multivariate analysis hr (95% ci) p value* age 0.93(0.87-1.00) 0.59 comorbidities (no/yes) 1.76(0.23-13.0) 0.57 stage (metastatic/non-metastatic) 0.07(0.007-0.74) 0.02 blood groups (a-b-o) 0.80 o reference a 0.42(0.07-2.34) 0.32 b 0.76(0.15-3.90) 0.74 ab 0.99(0.00--) 0.99 rh group (negative/positive) 1.66(0.25-10.7) 0.59 *binary logistic regression. discussion it is known that patients diagnosed with a malignant disease are predisposed to infections due to immunosuppression arising from comorbidities and treatment they receive. during the covid-19 pandemic, many unknown factors have become subjects of research, including, among other things, how and to what degree susceptible populations, such as those seemedj 2021, vol 5, no. 2 blood group and covid-19 in patients with malignant disease 6 southeastern european medical journal, 2021; 5(2) suffering from malignant diseases, are affected by the pandemic and what the factors affecting disease transmission and mortality are. this study aimed to investigate the risk factors affecting the transmission of and mortality from covid-19 in patients diagnosed with a malignant disease. the risk factors analysed included abo blood groups, gender, age, type of malignant disease, type of anti-tumoral agents, comorbidities and stage. of 1,256 patients who were examined and treated at our clinic and whose blood groups could be determined exactly, 5.7% (n=72) were diagnosed with covid-19 during the pandemic. since the pandemic period is a dynamic period, variability in this rate across regions and an increase over time are expected. previous studies conducted on patients diagnosed with a malignant disease have reported that advanced age, presence of comorbidities and receiving chemotherapy as opposed to other antitumor agents increase covid-19-related mortality (1214). in the evaluation of factors affecting mortality in patients infected with covid-19 in this study, age, gender, presence of comorbidities and treatment received by the patients (chemotherapy, immunotherapy, tki, hormonal therapy) were found to not influence disease-related mortality. covid-19 is thought to be associated with a poorer course in lung cancers. however, in this study, the diagnosis of primary malignancy did not have a significant correlation with mortality. however, metastatic stage was found to be an independent risk factor predicting covid-19 infection-related mortality (mortality rate of 26% in metastatic patients, 3.8% in non-metastatic patients (hr: 0.07, 95% ci (0.007-0.74), (p=0.02)). this can be explained by damage to natural barriers caused by cancer invasion and the patients’ relative vulnerability to secondary infections. abo blood group antigens are expressed in various tissues and cells, such as the epithelial, platelet, vascular endothelial cells and neurons. it is suggested that this system affects the spread of many pathogens. abo antibodies are also a component of the innate immune system (15,16). previous studies have found that susceptibility to viral infections is associated with abo blood group. for example, susceptibility to norwalk virus and hepatitis b was reported to vary depending on abo blood type (17,18). furthermore, individuals with o blood group were reported to have a lower likelihood of contracting sars coronavirus (19). do differences in blood group antigen expressions influence the risk of covid19 transmission and covid-19-related mortality? multiple epidemiological studies have been performed on this topic. some studies have found that the number of patients with type-a blood was higher among patients infected with sars-cov-2 compared with healthy controls, while the number of those with o blood group was much lower. some studies have also identified a correlation with the risk of mortality (9,20-22). in the study conducted by zhao j. and colleagues, it was reported that a blood group was associated with a predisposition to covid19 infection, whereas the rate of infection was lower and the course of infection milder in o blood type individuals (21). in the study by solmaz i. and colleagues conducted on a healthy population, covid-19-positivity was reported to be higher in a blood group and lower in o blood group. however, it was stated that the need for intensive care did not vary across the groups (22). in another study, arac e. and colleagues reported that there were no significant differences across the abo blood groups, but that a significant difference existed within the rh system, with rh positivity predisposing persons to covid-19 infection and rh negativity exerting protective effects against it (23). the present study did not include a healthy population. among the patients diagnosed with a malignant disease, those who did not become infected with covid-19 were considered the control group and those who became infected with covid-19 were considered the case group. a comparison of the group that contracted covid-19 and the group that did not revealed no significant differences in terms of abo blood groups (p=0.39). moreover, it was found that abo blood groups did not affect mortality in patients who became infected with covid-19 (p=0.83). similarly, it was also found that rh antigen positivity was not a factor affecting covid-19 transmission (p=0.19) and associated mortality (p=0.78). in this study, seemedj 2021, vol 5, no. 2 blood group and covid-19 in patients with malignant disease 7 southeastern european medical journal, 2021; 5(2) abo/rh blood groups were evaluated with regard to susceptibility to covid-19 infection and the related risk of mortality. the results of this study showed that covid-19 and abo/rh blood groups did not have a statistically significant correlation in terms of the risk of transmission or the risk of mortality. however, metastatic malignant disease was an independent risk factor for covid-19-related death. limitations of this study include its singlecentre, retrospective design and heterogeneity of the study population. conclusion this study found that metastatic malignant diseases increased covid-19-related mortality, while abo blood groups and the rh antigen status had no correlation with the risk of covid19 transmission and covid-19-related mortality. according to the results of this study, patients suffering from a metastatic malignant disease represent a high risk group for covid-19 and should be treated using all necessary precautions. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare references 1. kuderer nm, choueiri tk, shah dp, et al on behalf of the covid-19 and cancer consortium. clinical impact of covid-19 on patients with cancer (ccc19): a cohort study. lancet. 2020; 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294(7):794. cheng, yufeng [corrected to cheng, yunfeng]. pmid: 15784866. 20. li j, wang x, chen j, cai y, deng a, yang m. association between abo blood groups and risk of sars-cov-2 pneumonia. br j haematol. 2020; 190(1):24-27. doi: 10.1111/bjh.16797. epub 2020 may 26. pmid: 32379894; pmcid: pmc7267665. 21. zhao j, yang y, huang h, li d, gu d, lu x, zhang z, liu l, liu t, liu y, he y, sun b, wei m, yang g, wang x, zhang l, zhou x, xing m, wang pg. relationship between the abo blood group and the coronavirus disease 2019 (covid-19) susceptibility. clin infect dis. 2021; 73(2):328-331. doi: 10.1093/cid/ciaa1150. pmid: 32750119; pmcid: pmc7454371. 22. solmaz i̇, araç s. abo blood groups in covid19 patients; cross-sectional study. int j clin pract. 2021; 75(4):e13927. doi: 10.1111/ijcp.13927. epub 2020 dec 19. pmid: 33296536; pmcid: pmc7883261. 23. arac e, solmaz i, akkoc h, donmezdil s, karahan z, kaya s, mertsoy y, yildirim ms, ekin n, arac s, demir c. association between the rh blood group and the covid-19 susceptibility. international journal of hematology and oncology. 2020 30. 81-86. 10.4999/uhod.204247. 1 author contribution. acquisition of data: ebinç s, oruç z, kalkan z, urakçı z, kaplan ma, küçüköner m, işıkdoğan a administrative, technical or logistic support: ebinç s, oruç z, kalkan z, urakçı z, kaplan ma, küçüköner m, işıkdoğan a analysis and interpretation of data: ebinç s, oruç z, kalkan z, urakçı z, kaplan ma, küçüköner m, işıkdoğan a conception and design: ebinç s, oruç z, kalkan z, urakçı z, kaplan ma, küçüköner m, işıkdoğan a critical revision of the article for important intellectual content: ebinç s, oruç z, kalkan z, urakçı z, kaplan ma, küçüköner m, işıkdoğan a drafting of the article: ebinç s, oruç z, kalkan z, urakçı z, kaplan ma, küçüköner m, işıkdoğan a final approval of the article: ebinç s, oruç z, kalkan z, urakçı z, kaplan ma, küçüköner m, işıkdoğan a obtaining funding: ebinç s, oruç z, kalkan z, urakçı z, kaplan ma, küçüköner m, işıkdoğan a provision of study materials or patients: ebinç s, oruç z, kalkan z, urakçı z, kaplan ma, küçüköner m, işıkdoğan a statistical expertise (statistical analysis of data): ebinç s, oruç z, kalkan z, urakçı z, kaplan ma, küçüköner m, işıkdoğan a seemedj 2021, vol 5, no. 1 life quality in diabetes mellitus 75 southeastern european medical journal, 2021; 5(1) original article quality of life assessment in type 2 diabetes patients with cardiovascular and/or diabetic complications 1 semir mehović 1*, slobodan janković 2, zana tafi 3 1 public institution “apoteke sarajevo”, kranjčevićeva 29, sarajevo, bosnia and herzegovina 2 faculty of medical sciences, university of kragujevac, kragujevac, serbia 3 institute for occupational medicine, sarajevo, bosnia and herzegovina *corresponding author: semir mehović, semir.mehovic1989@gmail.com received: nov 12, 2020; revised version accepted: jan 12 2021; published: apr 28, 2021 keywords: quality of life, diabetic complications, type 2 diabetes mellitus abstract introduction: type 2 diabetes mellitus is a chronic disease that is causing enormous economic and social costs. it is characterized by many microvascular and macrovascular complications, such as heart attack, stroke, retinopathy, nephropathy, neuropathy, etc. such complications can cause severe limitations and decrease the quality of life. the objective of this study was to assess the effect of type 2 diabetes mellitus on the quality of life using the eq-5d-5l questionnaire, taking into account cardiovascular complications (heart attack, hospitalization due to angina pectoris, stroke, hospitalization due to heart insufficiency, transient ischemic attack, coronary revascularisation), complications of diabetes (microalbuminuria, renal failure, retinopathy, and neuropathy), and demographic characteristics (age, gender, body mass index, height, and weight). materials and methods: this cross-sectional study included 484 participants with type 2 diabetes mellitus. quality of life was estimated by the euroqol instrument eq-5d-5l and visual analogue scale (vas). the following complications related to type 2 diabetes were taken into account: heart attack, hospitalization due to angina pectoris, stroke, hospitalization due to heart insufficiency, transient ischemic attack, coronary revascularization, microalbuminuria, renal insufficiency, retinopathy, and neuropathy. results: the mean value of the eq index was 0.895, with the value of -0.59 as the lowest, and 1.0 as the highest quality of life of the study patients. multivariate linear regression model showed that heart attack, hospitalization due to unstable angina pectoris, retinopathy, and neuropathy significantly decreased the quality of life of the study participants (p<0.05). spearman's correlation showed that there was a significant correlation between age, height, duration of type 2 diabetes, body mass index, and the eq index (p<0.001). conclusion: the results suggest that type 2 diabetes complications, such as heart attack, neuropathy, retinopathy, and hospitalization due to unstable angina pectoris significantly decrease the quality of life of type 2 diabetes mellitus patients (t2dm). (mehović s, janković s, tafi z. quality of life assessment in type 2 diabetes patients with cardiovascular and/or diabetic complications. seemedj 2021; 5(1); 75-88) seemedj 2021, vol 5, no. 1 life quality in diabetes mellitus 76 southeastern european medical journal, 2021; 5(1) introduction there are two types of methods that can be used for measuring health utilities: direct and indirect. so far, several direct methods have been developed, such as: 1) standard gamble: reflects the assessment of an individual’s preference or valuation of their current qol by quantifying tolerance to risk toward an ultimate bad outcome as a gamble for achieving a perfect state of health (1); 2) time trade-off: individuals are asked to choose between living in their current impaired health state for 10 years or living for a shorter period with perfect health (2); and 3) visual analogue scale: introspective scaling with a rating scale from 0 to 100, where a patient has to choose one number on this scale that correctly reflects his/her quality of life (3). on the other hand, there are many indirect methods which have already been validated, and they can be generic (e.g. the eq-5d questionnaire) or specific for certain diseases (e.g. fact-g). indirect methods do not give immediate values for health utilities; rather, an algorithm must be developed for a transformation of answers to utility values (3). t2dm is recognized as one of the most common diseases worldwide, which affects every country and different groups of people, regardless of the demographic and socio-economic status (4). microvascular and macrovascular complications are almost inevitable during the course of the disease. the most common microvascular complications are diabetic nephropathy, neuropathy, and retinopathy, while the most common macrovascular complications are peripheral vascular disease, myocardial infarction, stroke, and congestive heart failure (5). quality of life assessment in patients with t2dm is important for the development of progressive simulation models used for a longterm assessment of the cost and effectiveness of interventions for the treatment of type 2 diabetes and prevention of accompanying complications. assessing the utility is the main prerequisite for determining the qaly and it is very important to include participants with a full spectrum of characteristics and health complications (retinopathy, neuropathy, nephropathy, etc.) (6). zhang et al. conducted a study in the united states of america (usa) on 7327 participants with t2dm. the average eq index (utility) in this study was 0.80. they concluded that participants without complications of type 2 diabetes, cardiovascular risk factors, and other comorbidities, who were not obese and who had an average income of over usd 40,000, had a better average eq index, reaching 0.92 (7). arifin et al. also concluded that t2dm patients with macro/microvascular complications have a lower qol (0.77-0.79) than those without such complications (0.80). also, if they have more than two complications, their qol is even lower (0.74) (8). pham et al. showed on 214 t2dm patients that those with diabetic retinopathy have a lower qol (0.92) compared to those who have diabetic heart disease (0.94) or nephropathy (0.93) (9). a study in japan showed that t2dm patients with more serious microvascular complications have a lower qol compared to those who have one or no complications. patients with proliferative retinopathy had a qol of 0.85, while those with pre-proliferative retinopathy had a better quality of life (0.93). also, those suffering from stage four nephropathy had a qol of 0.78, while the qol of those suffering from stage one nephropathy was as high as as 0.93 (10). there are also other studies showing that patients with complications of type 2 diabetes not only live shorter, but their quality of life is also lower (11,12). however, there is a paucity of studies using the eq-5d-5l questionnaire for a quality of life assessment in t2dm patients, and their quality estimates vary within a broad range from 0.74 to 0.92 (13-17). eq-5d-5l questionnaire is a relatively new preference-based instrument with lower ceiling effects and a better sensitivity compared to the eq-5d-3l. those two questionnaires are most commonly used in clinical and outcomes research (18). the aim of this study was to assess the effect of type 2 diabetes mellitus on the quality of life using the eq-5d-5l questionnaire, taking into account cardiovascular complications (heart attack, hospitalization due to angina pectoris, stroke, hospitalization due to heart insufficiency, transient ischemic attack, and coronary seemedj 2021, vol 5, no. 1 life quality in diabetes mellitus 77 southeastern european medical journal, 2021; 5(1) revascularisation), complications of diabetes (microalbuminuria, renal insufficiency, retinopathy, and neuropathy) and demographic characteristics (age, gender, body mass index, height, weight, smoking status, duration of diabetes, and education level). materials and methods study population this was a cross-sectional study, approved by the ethics committee of the public institution health center of sarajevo canton, which included 509 patients from bosnia and herzegovina. between december 2019 and june 2020, 361 patients in total were interviewed in person, while 148 patients were interviewed online. interviews in person were conducted at several health centers in sarajevo, such as the vrazova, omer maslić, and kumrovec health center, (urban areas) and ilijaš health centre (rural areas). after reviewing the answers, 484 participants were eligible for the final analysis, while 25 were excluded due to incompleteness. criteria for inclusion in the study were: diagnosis of t2dm in the patient file, age >18 years, and signed informed consent for participation in the study. criteria for non-inclusion or exclusion from the study were: persons under the age of 18, not signing the informed consent, pregnancy and/or a diagnosis of a major psychiatric disorder in the patient file. questionnaire in order to collect the patient information, a tripartite questionnaire was used, assessing the demographic/socio-economic characteristics (age, gender, height, weight, education level, duration of type 2 diabetes, and smoking status), quality of life by the eq-5d-5l questionnaire and by the visual analogue scale (vas), and cardiovascular and diabetic complications. the following events were taken into account, reflecting the complications of type 2 diabetes: heart attack, hospitalization because of unstable angina pectoris, stroke, hospitalization because of heart failure, transient ischemic attack, repeated coronary revascularization, microalbuminuria, renal insufficiency, neuropathy, and retinopathy. eq-5d-5l questionnaire the eq-5d-5l is a generic (19-23) questionnaire validated for the assessment of the quality of life of individuals with various health conditions. it assesses five dimensions: mobility, self-care, usual activities, pain/discomfort, and anxiety/depression. each dimension is ranked at five levels: no problems – 1, slight problems – 2, moderate problems – 3, severe problems – 4, and extreme problems – 5. after filling in the questionnaire, depending on the patients’ answers, the five dimensions are combined in a five-digit number that describes the patient’s health state. the five-digit code is further converted to an eq index using an automatic calculator (24) and the results from our study were checked using the guidelines from a validation study conducted in poland (25). since there was no local set of values that had been validated, poland was selected due to its cultural similarity with bosnia and herzegovina. the eq index ranged between -0.59 (the lowest qol) and 1 (the highest qol). the eq-vas scale was used together with the eq-5d-5l questionnaire (26). a validated croatian version of eq-5d-5l was used in our study, since the official language of bosnia and herzegovina is the bosnian/croatian/serbian (bhs) language. a laptop/desktop version of the questionnaire was obtained from euroqol. statistical analysis the results were described by frequencies and proportions for categorical values, and by the arithmetic mean and standard deviation for continuous values (or by the median with interquartile range, if the data were not normally distributed). normality of data distribution was tested by the shapiro wilk and kolmogorovsmirnov tests. nonparametric tests, such as the mann-whitney u test, the kruskal-wallis test, and non-parametric anova, were used for comparison of the study groups. impact of health complications, demographic seemedj 2021, vol 5, no. 1 life quality in diabetes mellitus 78 southeastern european medical journal, 2021; 5(1) characteristics, and health state were assessed by a multivariate linear regression model after confirming that the following assumptions were satisfied: linear relationship, independence, homoscedasticity and normality (27). results with α ≤ 0.05 or within the 95% confidence interval were considered statistically significant. spss for windows (version 21.0, spss inc, chicago, illinois, usa) and microsoft excel (version 11.0, microsoft corporation, redmond, wa, usa) were used for statistical analysis. results the final analysis included 484 participants (28.7% men and 71.3% women). mean duration of t2dm was 8.18 ± 7.08 years and the majority of participants were taking only oral antidiabetic drugs (55.8%) or oral drugs in combination with subcutaneous medication (41.7%). other demographic characteristics of the study participants are shown in table 1. more detailed results are shown in supplement 1. table 1. main demographic/socioeconomic characteristics of study participants (n=484). characteristic minimum maximum mean median std. deviation age (years) 20 92 52.68 53 12.11 body weight (kg) 47 186 89.00 88 18.27 height (m) 1.45 2.00 1.70 1.69 0.08 duration of type 2 diabetes (years) 0.02 40 8.18 6.00 7.08 body mass index (bmi) (kg/m2) 18.36 55.54 30.70 30.12 5.68 characteristic n (%) men women education level primary school high school education associate degree bachelor’s degree master’s/doctor’s degree smoking status yes, active smoker no, never smoked no, stopped smoking type of therapy oral oral + subcutaneous subcutaneous 139 (28.7) 345 (71.3) 17 (3.5) 275 (56.8) 61 (12.6) 124 (25.6) 7 (1.4) 188 (38.8) 153 (31.6) 143 (29.5) 270 (55.8) 202 (41.7) 12 (2.5) the majority of participants did not have problems with mobility (61%), but 1% of them had extreme problems. also, most of them did not have problems with self-care (79.5%) and usual activities (62.8%). on the other hand, a high percentage of participants had slight or moderate problems with pain/discomfort and anxiety/depression. more details about the participants’ answers are shown in table 2.. seemedj 2021, vol 5, no. 1 life quality in diabetes mellitus 79 southeastern european medical journal, 2021; 5(1) table 2. distribution of the euroqol 5d-5l answers given by study participants (n=484) according to five dimensions of this questionnaire no problems slight problems moderate problems severe problems extreme problems mobility 295 (61%) 76 (15.7%) 85 (17.6%) 23 (4.8%) 5 (1%) self-care 385 (79.5%) 45 (9.3%) 42 (8.7%) 8 (1.7%) 4 (0.8%) usual activities 304 (62.8%) 86 (17.8%) 73 (15.1%) 15 (3.1%) 6 (1.2%) pain/discomfort 214 (44.2%) 141 (29.1%) 100 (20.7%) 25 (5.2%) 4 (0.8%) anxiety/depression 201 (41.5%) 137 (28.3%) 109 (22.5%) 31 (6.4%) 6 (1.2%) using the eq-vas scale, the participants indicated how they felt at the moment of the interview. the minimum value was 5 and maximum was 100, with 70.40 (sd 20.49) points as the average value. the lowest eq index was 0.59 and the highest one was 1.0, with 0.895 (sd 0.183) on average (table 3). table 3. scores on eq vas scale and eq index of study participants (n=484) minimum maximum mean median std. deviation kolmogorov-smirnov eq vas scale 5 100 70.40 75 20.49 p = 0.000 eq index -0.59 1.00 0.895 0.952 0.183 p = 0.000 most of the participants did not have any cardiovascular/diabetic complications (46.90%) or had just one (23.97%). the most common complications were neuropathy (31.4%) and retinopathy (28.9%), while microalbuminuria (2.7%) and stroke (3.1%) were the rarest. the most common events occurring due to cardiovascular complications were hospitalization due to unstable angina pectoris (8.3%) or heart attack (8.1%). renal failure was reported in 9.1% of the cases. more details can be seen in supplements 2 and 3. spearman's correlation showed that there was a significant correlation between age, height, duration of type 2 diabetes and bmi (p<0.001) with the eq index. there was a negative correlation between bmi, age and duration of type 2 diabetes and the eq index (table 4). table 4. correlation between demographic characteristics and eq index of study participants (n=484) spearman's correlation coefficient p* height 0.223 0.000 body weight 0.019 0.675 bmi -0.106 0.000 age -0.254 0.000 duration of type 2 diabetes -0.228 0.000 we analysed whether gender (mann-whitney u test) and level of education/smoking status (kruskal-wallis test) influenced the eq index values. the results showed a significant influence of gender (u=19526.500, p=0.001) and level of education (kruskal-wallis h 9.949, seemedj 2021, vol 5, no. 1 life quality in diabetes mellitus 80 southeastern european medical journal, 2021; 5(1) p=0.041) on the eq index. smoking status did not affect the quality of life (kruskal-wallis h 1.082, p=0.582). multivariate linear regression was used to determine the effects of study variables on the quality of life. the eq index was appointed as a dependent variable, and gender, age, level of education, weight, height, bmi, duration of type 2 diabetes, and cardiovascular/diabetic complications were used as independent variables in the analysis. results showed that four complications – neuropathy, heart attack, stroke, and hospitalization because of unstable angina pectoris – had the greatest impact on the quality of life. these four dependent variables explained 49.3% of model variance (r=0.493, f=34.550, p=4.98*10-25) (table 5). table 5. effects of certain diabetic complications on the qol of study participants (n=484): results of multivariate linear regression model b sig. 95% confidence interval for b lower bound upper bound neuropathy 0.675 0.000 0.610 0.739 neuropathy heart attack 0.115 0.178 0.000 0.000 0.079 0.119 0.150 0.237 neuropathy heart attack stroke 0.103 0.174 0.263 0.000 0.000 0.000 0.069 0.117 0.176 0.138 0.230 0.350 neuropathy heart attack stroke unstable angina 0.097 0.142 0.249 0.081 0.000 0.000 0.000 0.017 0.062 0.080 0.162 0.147 0.944 0.807 0.970 0.776 discussion this cross-sectional study conducted in bosnia and herzegovina investigated the association between type 2 diabetes, its complications and the qol using the eq-5d-5l questionnaire. eq5d-5l is an easy-to-understand and validated generic questionnaire for a qol assessment (28). bmi, age, and duration of t2dm showed a negative correlation with the qol, meaning that older patients, with a higher bmi and longer duration of illness had a lower qol. the results also showed that most of the participants did not have problems with mobility or self-care, but they had problems with pain/discomfort and anxiety/depression, which is similar to previous studies. regarding t2dm patients, luk et al. reported that more patients had problems with pain/discomfort (24.8%) and anxiety/depression (20.3%) than with other dimensions of the eq-5d5l questionnaire (29). in several other studies, it has been shown that pain/discomfort was the most affected domain in patients (30-32). such results may be due to the fact that the most common t2dm complication was neuropathy, which may cause pain/discomfort. also, t2dm can change mood and lower self-esteem, leading to anxiety and depression (33). a study by parik and patel showed that anxiety and depression have a stronger impact on younger t2dm patients (34). a code-2 study on 1,371 t2dm participants showed that anxiety and depression were increased at a younger age and seemedj 2021, vol 5, no. 1 life quality in diabetes mellitus 81 southeastern european medical journal, 2021; 5(1) then decreased with age (35). auslli et al. suggested that patients with t2dm should be advised carefully about the importance of foot care and exercise. they concluded that physical exercise is a very important determinant of positive clinical outcomes of t2dm and it is associated with better hba1c levels, a lower bmi, fewer diabetic complications and a higher qol (36). our results also showed that the qol is dependent on gender, age, and education level, where females, older participants and those with a lower level of education had a lower qol. smoking status and weight did not have a significant impact on the qol. a study in korea showed that age is an important factor of the qol of patients with t2dm and that younger participants had a better qol, most likely because they had t2dm for a shorter period of time and fewer health-related complications (37). stojanović et al. also showed that lower level of education was highly related to lower qol. possible mechanisms include insufficient access to healthcare services, unhealthy habits, poor mental health, and higher frequency of complications (38). a study in denmark, involving 2419 patients with t2dm, showed that poor socioeconomic status, age, female gender, presence of comorbidity, poor glycemic control and lower level of education were associated with depressive episodes and lower qol of t2dm participants (39). there have been many other studies showing that these factors are strong determinants of the qol (37-50). most of the participants did not have any diabetic complications (46.90%). among those who had some of the complications, the most common were neuropathy (31.4%) and retinopathy (28.9%), while microalbuminuria and stroke were the rarest. data from the american diabetes association shows that 75-80% of adults with diagnosed dm will ultimately die from cardiovascular disease due to chronic macrovascular complications (41). microvascular and macrovascular complications of t2dm put a great burden on every healthcare system, even in developed countries like the usa. however, this is not the only problem. many studies have shown that patients with t2dm who have one or more diabetic complications also have a significantly lower qol compared to those who do not have t2dm-related complications (4246). in our study, the values of the vas scale varied from 5 to 100, with 70.40 as the average value. after converting the scores to the eq index, the lowest value was -0.59 and the highest was 1.0., with 0.895 as the average value. according to mlitt et al., a negative index is possible when researchers use the eq-5d-5l questionnaire (40). it suggests that the health state of these participants is deemed to be worse than death. the vas scale score can vary between different group of participants. a norwegian study of t2dm patients showed a mean eq index of 0.85 (51), while an iranian study reported an even lower average value (0.70) (52). a multivariate linear regression model based on our data showed that factors such as neuropathy, heart attack, stroke, and hospitalization due to angina pectoris are strong predictors of the qol, which is a finding congruent with the results of previous studies analysing patients with diabetes type 2. a univariate and multivariate regression analysis by stojanović et al. also showed that angina pectoris, heart failure, diabetic retinopathy, and diabetic nephropathy are factors that impact the qol of t2dm patients the most (38). a study in china, which was conducted on 1,275 patients with t1dm, showed that the presence of any of the four major diabetic complications (heart disease, stroke, end-stage renal disease, sightthreatening diabetic retinopathy) have a significant impact on the qol (48). the united kingdom prospective diabetes study showed that utilities are significantly reduced in t2dm patients who have serious complications, like end-stage renal disease, blindness, stroke or heart attack (50). there is a plethora of other studies showing that t2dm patients with complications have a decreased qol compared to t2dm patients without such complications (12,49,51,53,54). our study has certain limitations. firstly, the eq-5d-5l questionnaire was not previously validated in bosnia and herzegovina. therefore, seemedj 2021, vol 5, no. 1 life quality in diabetes mellitus 82 southeastern european medical journal, 2021; 5(1) the transformation of raw scores to the eq index had to be based on the results from another population that is as similar as possible to the bosnian population in terms of culture (the polish population), which still creates a certain potential for bias. secondly, the sample used in the study was not ideally balanced in regard to frequency and severity of type 2 diabetes complications, which could have led to an overestimate of the eq scores. conclusions our study gave an overview of average eq index values in the population of diabetes type 2 patients from bosnia and herzegovina, and showed a prevailing influence of diabetic complications on the quality of life, which could be used for further pharmacoeconomic analyses. demographic characteristics, such as the bmi, age and duration of t2dm were negatively correlated with the qol, meaning that patients with a higher bmi, who are older and have suffered from the illness for a longer period of time, had a lower qol. neuropathy, heart attack, stroke, and hospitalization due to unstable angina had the greatest impact on the qol in t2dm patients. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. slaughter kb, meyer eg, bambhroliya ab, meeks jr, ahmed w, bowry r, behrouz r, mir o, begley c, tyson je, miller c, warach s, grotta jc, mccullough ld, savitz si, s vahidy fs. direct assessment of health utilities using the standard gamble among patients with primary intracerebral hemorrhage. circ cardiovasc qual outcomes. 2019; 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8:18. doi: 10.1186/14777525-8-18 52. javanbakht m, abolhasani f, mashayekhi a, baradaran hr, jahanhirinoudeh y. health related quality of life in patients with type 2 diabetes mellitus in iran: a national survey. plos one. 2012; 7: e44526. 53. khunkaew s, fernandez r, sim j. demographic and clinical predictors of healthrelated quality of life among people with type 2 diabetes mellitus living in northern thailand: a cross-sectional study. health qual of life outcomes. 2019; 17(1):177. doi: 10.1186/s12955019-1246-2 54. clarke pm, hayes aj, glasziou pg, scott r, simes j, keech ac. using the eq-5d index score as a predictor of outcomes in patients with type 2 diabetes. med care. 2009; 47:61-68. seemedj 2021, vol 5, no. 1 life quality in diabetes mellitus 86 southeastern european medical journal, 2021; 5(1) supplements supplement 1. eq index mean values (n=484) number of patients (n=484) eq index: mean (sd) gender male female 139 345 0.894 (0.250) 0.895 (0.148) age (years) 18-25 26-35 36-45 46-55 56-65 66+ 7 32 101 151 115 78 0.940 (0.838) 0.926 (0.106) 0.934 (0.100) 0.914 (0.134) 0.888 (0.189) 0.803 (0.304) education level primary school high school education associate degree bachelor’s degree master’s/doctor’s degree 17 275 61 124 7 0.810 (0.181) 0.902 (0.165) 0.882 (0.234) 0.925 (0.126) 0.798 (0.216) bmi (kg/m2) 18-24.99 25-29.99 30-34.99 >35 95 134 161 94 0.884 (0.257) 0.880 (0.216) 0.920 (0.115) 0.885 (0.128) duration of diabetes (years) 0.01-2.99 3.00-5.99 6.00-8.99 9.00-14.99 > 15 121 107 67 119 70 0.945 (0.077) 0.888 (0.186) 0.910 (0.121) 0.904 (0.107) 0.788 (0.349) smoking status yes, active smoker no, never smoked no, stopped smoking 188 153 143 0.897 (0.175) 0.890 (0.164) 0.889 (0.214) seemedj 2021, vol 5, no. 1 life quality in diabetes mellitus 87 southeastern european medical journal, 2021; 5(1) supplement 2. type 2 diabetes related complications complication n (%) heart attack yes no hospitalization due to unstable angina pectoris yes no stroke yes no hospitalization due to heart failure yes no transient ischemic attack yes no coronary revascularisation yes no microalbuminuria yes no renal insufficiency yes no retinopathy yes no neuropathy yes no 39 (8.1) 445 (91.9) 40 (8.3) 444 (91.7) 15 (3.1) 469 (96.9) 21 (4.3) 463 (95.7) 27 (5.6) 457 (94.4) 35 (7.2) 449 (92.8) 13 (2.7) 471 (97.3) 44 (9.1) 440 (90.9) 140 (28.9) 344 (71.1) 152 (31.4) 332 (68.6) seemedj 2021, vol 5, no. 1 life quality in diabetes mellitus 88 southeastern european medical journal, 2021; 5(1) supplement 3. number of complications discovered in the study population number of complications n (%) without complications 227 (46.90) one complication 116 (23.97) two complications 75 (15.50) three complications 26 (5.37) four complications 26 (5.37) five complications 9 (1.86) six complications 3 (0.62) seven complications 1 (0.21) eight complications 1 (0.21) author contribution. mehović s., janković s., and zana s. conceived the study design and participated in data collection. mehović s. and janković s. led the data analysis and interpretation. all authors approved the final manuscript. seemedj 2020, vol 4, no. 2 pathohistological diagnosis of adrenal tumors 108 southeastern european medical journal, 2020; 4(2) original article pathohistological diagnosis of adrenal tumors: experience of a single center 1 ivan lekić 1, veronika banović 1, ksenija marjanović 1,2, borna kovačić 1,3, ivan feldi 1,4, tatjana bačun *1,5 1 faculty of medicine osijek, josip juraj strossmayer university osijek, croatia 2 department of pathology and forensic medicine, university hospital center osijek, croatia 3 surgery clinic, department of abdominal surgery, university hospital center osijek, croatia 4 department of internal medicine, našice general hospital, našice, croatia 5 clinic for internal medicine, department of endocrinology, university hospital center osijek, croatia *corresponding author: tatjana bačun, tbacun@gmail.com received: apr 4, 2020; revised version accepted: sep 28, 2020; published: nov 12, 2020 keywords: histology, adrenal gland, adrenal tumor, adrenocortical adenoma, myelolipoma abstract introduction: to investigate adrenal tumors of patients operated on at the university hospital center osijek from 2016 to 2019 for the purpose of examining the location and histopathological findings of the tumors and determining whether there is a difference related to that in terms of the age and sex of the subjects. subjects and methods: this was a cross-sectional study with historical data analysis. the subjects were patients of both sexes (n=23) diagnosed with adrenal tumors (n=23) who underwent surgery at the university hospital center osijek in the period from 2016 to 2019. the archive was used in the university hospital center osijek. results: adrenal tumors were more often unilateral than bilateral. no differences were found in the localization of tumors of the right and left adrenal glands. benign tumors were more common than malignant tumors and were the most common adenoma. seventeen women underwent surgery and were 10 years older on average (61.6 years). there were no significant differences regarding tumor occurrence with respect to the patients’ age. benign tumors were more common in women, but the difference was not statistically significant. regarding histopathological findings, women were more likely to have adenoma (n=10) and hyperplasia (n=4), while men had other benign tumors (n=3) and metastatic tumors (n=1). however, the difference was not statistically significant. conclusion: in patients who have undergone surgery for adrenal tumors, there were more unilateral tumors compared to bilateral ones, but there was no difference in tumor localization (left and right). benign tumors were more common, the most common of which were adenomas. there were no significant differences with respect to age and sex. (lekić i, banović v, marjanović k, kovačić b, feldi i, bačun t. pathohistological diagnosis of adrenal tumors: experience of a single center. seemedj 2020; 4(2); 108-112) seemedj 2020, vol 4, no. 2 pathohistological diagnosis of adrenal tumors 109 southeastern european medical journal, 2020; 4(2) introduction the adrenal glands are bilateral glands located above each kidney, which typically weigh about 5 grams, but may be much larger in patients with chronic disease due to increased stimulation. the right adrenal gland is pyramid-shaped, while the left is crescent-shaped. the adrenal cortex in adults accounts for about 90% of the weight of the glands and there are three zones of the cortex: the glomerulosa zone, the fasciculata zone, and the reticularis zone. the neoplasms of the adrenal cortex can be benign (usually adenomas), malignant (cancers) and metastatic tumors. furthermore, adrenal tumors can be hormone-active and inactive. both functional and non-functional tumors occur more frequently in women than in men between the ages of 40 and 60 (1). adenomas of the adrenal cortex are the most commonly benign lesions of the adrenal glands and the most common lesions in general (2). adrenocortical cancers are extremely rare, with an incidence rate of one to two cases per million people per year, and as many as 15% of adrenal cancers are detected by chance. they have a bimodal distribution, that is, they appear in the first and fourth decade of life and are estimated to be functional in as many as 80% of cases, resulting in a characteristic clinical picture and in 55-60% of cases involving the female sex (3). the adrenal tumors (adenomas and hyperplasia) are round, of well-circumscribed margins and a homogeneous structure. these tumors have visible separation from the surrounding tissues and do not branch into surrounding structures. this study investigates the localization and pathohistological findings of adrenal neoplasms, as well as differences in terms of age and sex in subjects with adrenal tumors who underwent surgery at the university hospital center osijek from 2016 to 2019. patients and methods the study involved an analysis of the medical records on patients diagnosed with adrenal tumors who underwent surgery at the department of abdominal surgery of the university hospital center osijek from 2016 to 2019, which are kept by the department of abdominal surgery of the university hospital center osijek and the clinical institute of pathology and forensic medicine. the data collected comprised: the patients’ age and sex, diagnosis, tumor localization, pathohistological findings and the degree of pathohistological differentiation according to weiss criteria. adrenal tumor samples were analyzed morphologically using an olympus® cx40 microscope on existing archival histological slides stained with the standard hematoxylin and eosin stain. the subjects were patients of both sexes diagnosed with adrenal tumors who underwent surgery at the university hospital center osijek in the period from 2016 to 2019. the sample consisted of a total of 23 patients (n = 23). samples of adrenal tumors were examined histologically at the clinical institute of pathology and forensic medicine, university hospital center osijek, which determined the degree of pathohistological differentiation according to weiss criteria. the study used the medical records on patients diagnosed with adrenal tumors who were operated on at the department of abdominal surgery of the university hospital center osijek from 2016 to 2019, which are kept by the department of abdominal surgery of the university hospital center osijek and the clinical institute of pathology and forensic medicine. the analysis was performed on existing archival histological slides using standard (hematoxylin and eosin) staining (n=23). approval of the institutional ethical committee for this study was obtained from faculty of medicine osijek. statistical analysis in statistical analysis three tests were conducted. fisher’s exact test, anova test and student’s t-test. fisher’s exact test was used for statistical analysis of unilateral or bilateral adrenal neoplasms, malignancy between left or right neoplasms, pathological differences on adrenal neoplasms between sexes, localization of adrenal neoplasms between sexes. anova seemedj 2020, vol 4, no. 2 pathohistological diagnosis of adrenal tumors 110 southeastern european medical journal, 2020; 4(2) test was used for statistical analysis for pathological diagnosis and age. student’s t-test was used for statistical analysis for age and sex and for age and size of adrenal neoplasms. p<0.05 was set as a level of significance. results patients who have undergone surgery on for adrenal tumors at the university hospital center osijek from 2016 to 2019 are shown. during this period, 23 patients were surgically treated. tumors were more often unilateral (n = 18) than bilateral (n = 5). no differences were found between tumor localization in the left (n = 9) and right (n = 9) adrenal glands. in this study, it was observed that benign neoplasms had a higher incidence (n = 22) than malignant neoplasms (n = 1). adrenal adenomas (n = 12) and hyperplasia (n = 4) have the highest incidence of benign neoplasms, while only metastatic renal cancer was observed in malignant neoplasms (n = 1). the average age of the subjects was 58.9 (sd 11.6). the average age of men was 51.2 (sd 16.6). the average age of women was 61.6 (sd 8.3). it was observed that tumors occur in women at a later age, but the difference was not statistically significant (student's t-test, p = 0.189). the mean age of patients with unilateral tumors was 59.1 (sd 12.7) and with bilateral tumors 58.4 (sd 7.16). there was no statistically significant difference in adrenal tumor distribution in respect to age (data not shown) (student's t-test, p = 0.914). the average age of patients with tumors of the left adrenal gland was 63.6 (sd 9.96) and of the right adrenal gland 54.6 (sd 14.13), which was similar (anova, p = 0.267). the average age of individuals with adenoma was 60.3 (sd 9.17), with hyperplasia 61.5 (sd 6.14) and with other benign tumors 54.7 (sd 18.77). there was no statistically significant difference in respect to age and phd of the tumor (anova, p = 0.777). benign tumors were more common in women, but the difference was not statistically significant (fisher's exact test, p = 0.261). according to the histopathological findings of adrenal tumors, women had more frequent adenomas and hyperplasia, while benign tumors and metastases were more frequent in men, but the difference was not statistically significant (fisher's exact test, p = 0.110). the average tumor size was 4.87 cm (sd 1.95 cm). the average tumor size in men was 5.77 cm (sd 2.42). the average tumor size in women was 4.55 cm (sd 1.73), which was similar between men and women. the average adenoma size was 4.098 cm (sd 1.22), hyperplasia 4.18 (sd 0.96), other benign tumors 7.22 (sd 1.91). patients with unilateral tumors were slightly older than patients with bilateral tumors. the patients with tumors of the left adrenal gland were slightly older than the patients with tumors of the right adrenal gland. the patients with adenoma and adrenal hyperplasia were older than patients with other benign adrenal changes. there was no correlation between age and histopathological diagnosis of the tumor (data not shown). women with adrenal tumors who were surgically treated were older than men. men averaged 51.2 years of age (sd 16.6), while women averaged 61.6 years of age (sd 8.3), but the difference in age was not statistically significant. discussion the study is a review of archival medical records kept by the department of abdominal surgery of the university hospital center osijek and the clinical institute of pathology and forensic medicine from 2016 to 2019. the data were collected for 23 patients diagnosed with adrenal tumors who were operated on at the department of abdominal surgery of the university hospital center osijek. the data collected were analyzed to examine the existence of differences in localization (singlesided localization, left-right localization) and histopathological findings of tumors between subjects of both sexes. in our study, it was observed that unilateral neoplasms are more common than bilateral ones, which is consistent with the results of other studies (1,4,10,11). data on tumor localization vary considerably in different studies and their samples. mantero et al. and kasperlik-zaluska et seemedj 2020, vol 4, no. 2 pathohistological diagnosis of adrenal tumors 111 southeastern european medical journal, 2020; 4(2) al. showed in their studies that neoplasms in the right adrenal gland were more common, unlike bovio et al., who observed that tumors were more common in the left adrenal gland (3,5,6). in our study, neoplasms of the right and left adrenal glands were equally common, which is in agreement with a similar study by casola et al. (7). in our study, obtained data suggest that benign adrenal neoplasms are the most common neoplasms (figure 3), which is in agreement with a study by szolar et al. and cawood et al. (8.9). more than half of pathohistological diagnoses were adenomas, which is in line with other studies that cite adenomas as the most common pathohistological diagnosis (8,9). metastases are the most common malignant adrenal lesions and the second most common adrenal lesions in general (after adenoma) (2). the most common cancers that metastasize to the adrenal gland are cancers of the breast, lung, stomach, kidney and melanoma (10). in the case of multiple metastases, treatment is performed by methods other than solitary metastases only in the adrenal glands that are treated surgically (5). according to angeli a et al., women are more commonly affected by adrenal tumors (1,2), which, in addition to the statistical significance, is shown by the results of this study based on the data from the university hospital center osijek. the risk of malignancy increases in proportion to the size of the tumor (12,13). the best ratio of sensitivity to specificity is the tumor diameter of 4 cm (5). considering the limitations due to a small sample size used in our study, the value of this research is that it demonstrates our center's experience with histopathological findings of adrenal tumors. monitoring needs to be continued in order to obtain data over a longer period. adrenal tumors were more often unilateral than bilateral, with equal incidence of the tumors of the right and left side, more often benign, most often adenomas. there was no significant difference with respect to age. women had adenomas and hyperplasia more frequently, while other benign tumors and metastatic tumors were more frequent in men, but the difference was not statistically significant. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. i references 1. angeli a, osella g, alì a, terzolo m. adrenal incidentaloma: an overview of clinical and epidemiological data from the national italian study group. horm res 1997; 47:279. 2. iñiguez-ariza nm, kohlenberg jd, delivanis da, hartman rp, dean ds, thomas ma, shah mz, herndon j, mckenzie tj, arlt w, young jr wf, bancos i. clinical, biochemical, and radiological characteristics of a single-center retrospective cohort of 705 large adrenal tumors. mayo clin proc innov qual outcomes 2018; 2:30. 3. bovio s, cataldi a, reimondo g, sperone p, novello s, berruti a, borasio p, fava c, dogliotti l, scagliotti gv, angeli a, terzolo m. prevalence of adrenal incidentaloma in a contemporary computerized tomography series. j endocrinol invest 2006; 29:298. 4. barzon l, scaroni c, sonino n, fallo f, gregianin m, macri c, boscaro m. incidentally discovered adrenal tumors: endocrine and scintigraphic correlates. j clin endocrinol metab 1998; 83:55. 5. mantero f, terzolo m, arnaldi g, osella g, masini am, ali a, giovagnetti m, opocher g, angeli a. a survey on adrenal incidentaloma in italy. study group on adrenal tumors of the italian society of endocrinology. j clin endocrinol metab 2000; 85:637. seemedj 2020, vol 4, no. 2 pathohistological diagnosis of adrenal tumors 112 southeastern european medical journal, 2020; 4(2) 6. kasperlik-zeluska aa, rosłonowska e, słowinska-srzednicka j, migdalska b, jeske w, makowska a, snochowska h. incidentally discovered adrenal mass (incidentaloma): investigation and management of 208 patients. clin endocrinol (oxf) 1997; 46:29. 7. casola g, nicolet v, vansonnenberg e, et al. unsuspected pheochromocytoma: risk of blood-pressure alterations during percutaneous adrenal biopsy. radiology 1986; 159:733-735 8. szolar dh, korobkin m, reittner p, berghold a, bauernhofer t, trummer h, schoellnast h, preidler kw, samonigg h. adrenocortical carcinomas and adrenal pheochromocytomas: mass and enhancement loss evaluation at delayed contrast-enhanced ct. radiology 2005; 234:479. 9. cawood tj, hunt pj, o'shea d, cole d, soule s. recommended evaluation of adrenal incidentalomas is costly, has high false-positive rates and confers a risk of fatal cancer that is similar to the risk of the adrenal lesion becoming malignant; time for a rethink? eur j endocrinol 2009; 161:513. 10. young wf jr. clinical practice. the incidentally discovered adrenal mass. n engl j med 2007; 356:601. 11. young wf jr. management approaches to adrenal incidentalomas. a view from rochester, minnesota. endocrinol metab clin north am 2000; 29:159. 12. hamrahian ah, ioachimescu ag, remer em, motta-ramirez g, bogabathina h, levin hs, reddy s, gill is, siperstein a, bravo el. clinical utility of noncontrast computed tomography attenuation value (hounsfield units) to differentiate adrenal adenomas/hyperplasias from nonadenomas: cleveland clinic experience. j clin endocrinol metab 2005; 90:871. grumbach mm, biller bm, braunstein gd, et al. management of the clinically inapparent adrenal mass ("incidentaloma"). ann intern med 2003; 138:424. i author contribution. acquisition of data: lekić i, banović v, marjanović k, kovačić b, feldi i, bačun t administrative, technical or logistic support: lekić i, banović v, marjanović k, kovačić b, feldi i, bačun t analysis and interpretation of data: lekić i, banović v, marjanović k, kovačić b, feldi i, bačun t conception and design: lekić i, banović v, marjanović k, kovačić b, feldi i, bačun t critical revision of the article for important intellectual content: lekić i, banović v, marjanović k, kovačić b, feldi i, bačun t drafting of the article: lekić i, banović v, marjanović k, kovačić b, feldi i, bačun t final approval of the article: lekić i, banović v, marjanović k, kovačić b, feldi i, bačun t guarantor of the study: lekić i, banović v, marjanović k, kovačić b, feldi i, bačun t obtaining funding: lekić i, banović v, marjanović k, kovačić b, feldi i, bačun t provision of study materials or patients: lekić i, banović v, marjanović k, kovačić b, feldi i, bačun t statistical expertise: lekić i, banović v, marjanović k, kovačić b, feldi i, bačun t other: lekić i, banović v, marjanović k, kovačić b, feldi i, bačun t seemedj 2021, vol 5, no. 1 croatian cips 145 southeastern european medical journal, 2021; 5(1) review article translation of the clance impostor phenomenon scale into the croatian language 1 ivona čarapina zovko 1, jakov milić 2*, filip bartolomeo vucemilovic 3, nika jemrić 4, petra sulić 5, matea turudić 4, dominic vidović 4, dorotea jelovica 5, ivan padjen 6, 7, gordana ivanac 7, 8, vedrana ivić 9, ivana škrlec 10, zrinka biloglav 11 1 department of psychology, faculty of humanities and social sciences, university of mostar, bosnia and herzegovina 2 catholic faculty of theology, university of zagreb, zagreb, croatia 3 department of emergency medicine dubrovnik-neretva county, dubrovnik, croatia 4 general practitioner’s office md, marija petrovčić, zagreb, croatia 5 department of inclusive education and rehabilitation, faculty of education and rehabilitation sciences, university of zagreb, croatia 6 division of clinical immunology and rheumatology, department of internal medicine, university hospital centre zagreb, croatia 7 school of medicine, university of zagreb, croatia 8 department of diagnostic and interventional radiology, university hospital dubrava, zagreb, croatia 9 department of medical biology and genetics, faculty of medicine osijek, j.j. strossmayer university of osijek, croatia 10 department of biophysics, biology, and chemistry, faculty of dental medicine and health osijek, j. j. strossmayer university of osijek, croatia 11 department of medical statistics, epidemiology and medical informatics, school of public health andrija stampar, university of zagreb school of medicine, zagreb, croatia corresponding author: jakov milić, milic.jakov@gmail.com received: dec 17, 2020; revised version accepted: feb 14 2021; published: apr 28, 2021 keywords: croatian language, psychometrics, instruments, impostor syndrome, translation seemedj 2021, vol 5, no. 1 croatian cips 146 southeastern european medical journal, 2021; 5(1) introduction the psychological term impostor phenomenon (ip) can be identified using a combination of keywords and phrases, including spelling variations and synonyms, such as impostor phenomenon, impostorism, impostor syndrome, impostor, and perceived fraudulence (1). psychologists p. clance and s. imes coined the term in 1978 based on clinical observations and after clance published her book in 1985, the term attracted increasing attention. nowadays, the term is widely present due to its strong association with several personal and workrelated implications (2–4). impostorism is not a diagnosable mental illness or condition, yet a pervasive psychological experience of perceived intellectual and professional fraudulence. impostors doubt their accomplishments and have a persistent fear of being exposed as a fraud (5). despite adequate evidence of accomplishment, they feel selfdoubt and perceive themselves as unworthy of promotion, recognition and reward, convinced that they do not deserve the success (2). some characteristics indicative of a maladaptive personality style facilitate the vicious cycle of perceived inadequacy. impostors use increased efforts aimed towards achieving their goal to overcompensate for their frustration, but these well-known perfectionists and workaholics suffer from a considerable amount of pressure, anxiety, fear of failure and life dissatisfaction (3,6). impostorism occurs across different cultures and lifetime prevalence is as high as 70% (7–11). although clance and imes first noticed that this pattern was more common in females, subsequent studies yielded inconsistent results, indicating that the impostor experience was equally or even more common among men (3,6,11,12). it should be noted that it does not affect only highly successful people and that anyone could view themselves as impostors if they failed to internalize their success (13). the presence of impostorism has been studied among different occupations, such as college students (9,10,13), academics (11), physician assistants (14), marketing managers (15) and medical students (1,16). a highly competitive environment in the medical field, especially during the training period, probably contributes abstract introduction: clance impostor phenomenon scale (cips) is the most common and psychometrically sound instrument used to measure the impostor phenomenon. the aim of this study was to translate and make a cultural adaptation of the cips from english into croatian. materials and methods: the translation process included two independent forward translations, combination of the forward translations into one single translation, back-translation, back-translation review, pre-piloting and drafting of the final translation after several revisions and minor adjustments by a professional reviewer. results: we noticed no semantic differences when comparing the original and the back-translated versions of the cips. thus, the final translation was only slightly changed in comparison with the first version. conclusions: the version of the cips which was translated and culturally adapted into croatian represents a reliable translation ready to be used in croatia and bosnia and herzegovina. (čarapina zovko i, milić j, vucemilovic fb, jemrić n, sulić p, turudić m, vidović d, jelovica d, padjen ivanac g, ivić v, škrlec i, biloglav z. translation of the clance impostor phenomenon scale into the croatian language. seemedj 2021; 5(1); 145-156) seemedj 2021, vol 5, no. 1 croatian cips 147 southeastern european medical journal, 2021; 5(1) to the high prevalence of impostorism. however, this phenomenon, which affects nearly half of females and one-fourth of males among medical students and residents, is still under acknowledged by the healthcare community (17). initially, it was considered static, but subsequently, its’ quite dynamic nature was recognized, and the transition from preclinical to clinical training was described as particularly challenging (9–11). researchers have identified several factors contributing to the emergence of impostorism, such as perfectionism (2,18–20) and family environment (2,9,21,22). family dynamics and parenting style can impact the values and behaviours associated with children’s success, especially how the child will learn to cope with success and failure (23). general family characteristics were proposed as contributors to the reappearance of the impostor phenomenon: (i) the perception of the impostor that their talents are uncharacteristic compared to family members’ talents, (ii) family messages that convey the importance of intellectual ability and that success requires little effort, (iii) disagreement between feedback on the abilities and success of impostors stemming from the family and other sources and (iv) a lack of positive reinforcement or support. the tendency of impostors to reject positive feedback and uphold high standards for selfevaluation while remaining critical of their inability to achieve these standards shows consistency with perfectionism (23). regarding the ip and personality traits, neuroticism was identified as a strong predictor (23) and there are also positive associations with introversion (5), as opposed to conscientiousness and agreeableness (23). different definitions of the ip and various measurement scales were made for clinical and research purposes in order to quantify the characteristics of the impostor experience. the clance impostor phenomenon scale (cips), the most common and psychometrically sound instrument (2,24), was developed in 1985. besides the harvey impostor scale (11), there are also two other separate scales: the perceived fraudulence scale (pfs) (25) and the leary impostor scale (26). it should be noted that researchers defined the construct differently – harvey, clance, and kolligian and sternberg (3,11,25) suggested multidimensionality of impostorism, while leary et al. proposed a unidimensional definition (24,26). after years of researching this phenomenon, clance defined a typical impostor and created the cips that accurately measures the intensity an impostor could experience. this most commonly used instrument by researchers and practitioners has been validated in different settings. it contains items which address the fear of failure, attribution of success to luck, error, or charm, the desire to stand out, the feeling of having given others a false impression, the discounting of recognition from others, as well as the fear of evaluation, fear that successes cannot be repeated and the feeling that one is less capable than peers (2,24,27). this study aimed to translate and culturally adapt the cips to the croatian language, including translation and piloting of the questionnaire for students in croatia and bosnia and hercegovina. the croatian language is also one of the three official languages in bosnia and herzegovina, while the university of mostar is the only croatian-speaking university in the country. materials and methods the cips is a 20-item survey in which respondents rate their answers on a likert scale from 1 to 5, where the numbers have the following meanings: 1 – not at all true, 2 – rarely true, 3 – sometimes true, 4 – often true, or 5 – very true. for each question, respondents are advised to circle the number that best indicates how true the statement is for them and provide the first response that enters their mind, rather than dwelling on and overthinking each statement. the scores for each item add up to produce a total score, and higher scores indicate greater identification with the impostor syndrome (28). the translation algorithm for the cips (used with the authors’ permission) was selected according to the previously described methodology, which included two mandatory forward translations seemedj 2021, vol 5, no. 1 croatian cips 148 southeastern european medical journal, 2021; 5(1) and backward translation by a health professional (29). forward translations of the instrument were made by translators residing in the country and familiar with the field of health outcomes. a native english bilingual professional translator and a native croatian translator for the english language forwardtranslated the cips into croatian. once these versions were available, we discussed combined translation efforts at a consensus meeting. the statement “i’m often afraid that i may fail at a new assignment or undertaking even though i generally do well at what i attempt” was inadequately translated in the first forward translation, but the second one was more comparable with the original statement. regarding all other questions, the forward translations were similar to and consistent with the original version. subsequently, a bilingual native english-speaking medical professional, who was unaware of the original version, backtranslated the combined version of the translations into english. the objective of backtranslation was to detect errors in meaning and non-equivalence. we found no semantic or stylistic differences between the original and back-translation version of the cips. the algorithm of the translation protocol is presented in figure 1. figure 1. the algorithm of the translation protocol preliminary pilot testing was conducted to assess content accuracy and clarity of the language for both countries, croatia and bosnia and herzegovina. the questionnaire was administered to a convenient sample of 12 university students (5 males and 7 females, median age of 21, iqr of 21-35) from the university of zagreb. during pre-pilot testing, seemedj 2021, vol 5, no. 1 croatian cips 149 southeastern european medical journal, 2021; 5(1) the interviewer observed that the translations represented the source questionnaire effectively. the participants had suggestions for possible qualitative language improvements regarding some items, which were incorporated into the final version of the questionnaire. afterwards, the suggestions and small language differences were reviewed by a professional croatian language reviewer with 20 years of experience and they were incorporated into the croatian version of the cips. results in table 1, we provided a detailed presentation of the outcome of the translation process, which consists of the original version, two forward translations into croatian, a combined version of the translations and back-translation into english. the translators provided a translation that is as close to the original as possible and we did not find any significant changes in the meaning or style. when comparing the original english version and the back-translated version, we observed no relevant semantic differences. the only differences between the original and the back-translation were related to using different grammatical forms, which resulted in very similar meanings. these versions are comparable with the original english instrument in terms of content and accuracy, although the croatian version has been culturally adapted. table 1. steps for the cips translation into the croatian language original forward translation no. 1 forward translation no. 2 combined translation backward translation 1. i have often succeeded on a test or task even though i was afraid that i would not do well before i undertook the task. često bih uspješno napisao/la test ili izvršio/la zadatak iako sam se bojao/la da ga neću dobro napraviti prije nego sam ga započeo/la. često sam bio/la uspješan/na na testu iako sam se bojao/la da neću postići dobar rezultat prije početka rješavanja testa. često bih uspješno napisao/la test ili izvršio/la zadatak iako sam se prije početka bojao/la da ga neću dobro napraviti. i often do well on exams even though beforehand i was afraid of failing. 2. i can give the impression that i’m more competent than i really am. mogu odati dojam da sam sposobniji/a nego što zbilja jesam. ponekad se činim kompetentnijim/om nego što zapravo jesam. mogu odati dojam da sam sposobniji/a nego što zapravo jesam. i can give the impression that i’m more capable than i truly am. 3. i avoid evaluations if possible and have a dread of others evaluating me. izbjegavam procjene ako je moguće i grozim se toga da me drugi procjenjuju. ako mogu, izbjegavam evaluacije i strahujem od toga da me drugi procjenjuju na bilo koji način. ako mogu, izbjegavam procjene i strahujem od toga da me drugi procjenjuju. i avoid any comparison and i'm terrified of being judged by others. 4. when people praise me for something i’ve accomplished, i’m afraid i won’t be able to live up to their expectations of me in the future. kada me hvale za moja postignuća, bojim se da u budućnosti neću moći ispuniti njihova očekivanja. kada drugi pohvale moja postignuća, bojim se da neću ispuniti njihova očekivanja u budućnosti. kada me drugi hvale za moja postignuća, bojim se da neću moći ispuniti njihova očekivanja u budućnosti. when being praised for my achievements, i’m afraid of not being able to meet their expectations in the future. 5. i sometimes think i obtained my present position or gained my present ponekad mislim da sam došao/la do sadašnje pozicije ili trenutnog uspjeha ponekad mislim da sam trenutnu poziciju i trenutan uspjeh postigao/la ponekad mislim da sam postigao/la sadašnju poziciju ili sadašnji uspjeh jer i sometimes think that i made it to this position or point in life because i was at seemedj 2021, vol 5, no. 1 croatian cips 150 southeastern european medical journal, 2021; 5(1) success because i happened to be in the right place at the right time or knew the right people. jer sam bio/la na pravom mjestu u pravo vrijeme ili jer sam poznavao/la prave ljude. jer sam se našao/la na pravom mjestu u pravo vrijeme ili sam poznavao/la ljude koji su mi to omogućili. sam bio/la na pravom mjestu u pravo vrijeme ili sam poznavao/la prave ljude. the right place at the right time or due to knowing the right people. 6. i’m afraid people important to me may find out that i’m not as capable as they think i am. strahujem od toga da će ljudi koji su mi važni saznati da nisam sposoban/na koliko oni misle da jesam. bojim se da će ljudi koji su mi važni otkriti da nisam sposoban koliko su mislili. strahujem od toga da će meni važni ljudi otkriti da nisam sposoban/na koliko oni misle da jesam. i'm afraid that the people close to me will find out that i’m not as capable as they think i am. 7. i tend to remember the incidents in which i have not done my best more than those times i have done my best. sklon/a sam više pamtiti događaje u kojima nisam dao/la sve od sebe nego one kada sam dao/la sve od sebe. češće se prisjećam situacija u kojima sam bio/la neuspješan/na nego onih u kojima sam bio/la uspješan/na. sklon/a sam češće se prisjetiti događaja u kojima nisam dao/la sve od sebe nego onih kada sam dao/la sve od sebe. i’m more inclined to remember the times i didn’t give my best effort, compared to the times when i did. 8. i rarely do a project or task as well as i’d like to do it. rijetko kada projekt ili zadatak napravim onoliko dobro koliko bih volio/ljela. rijetko završim projekt ili zadatak zadovoljan/na odrađenim poslom. rijetko kada projekt ili zadatak napravim onoliko dobro koliko bih volio/ljela. rarely do i accomplish an assignment or project as well as i would of liked. 9. sometimes i feel or believe that my success in my life or in my job has been the result of some kind of error. ponekad se osjećam ili vjerujem da je uspjeh u mom životu ili na poslu posljedica nekog tipa greške. ponekad se osjećam ili mislim da je uspjeh u mom životu ili poslu rezultat nekakve pogreške. ponekad se osjećam ili vjerujem da je uspjeh u mom životu ili poslu posljedica nekakve pogreške. sometimes i feel or believe that success in my life or at work is the result of some type of mistake. 10. it’s hard for me to accept compliments or praise about my intelligence or accomplishments. teško prihvaćam komplimente ili hvalu na račun moje inteligencije ili uspjeha. teško prihvaćam komplimente ili pohvale vezane uz moju inteligenciju ili postignuća. teško prihvaćam komplimente ili pohvale na račun moje inteligencije ili postignuća. it’s difficult for me to accept compliments or praise based on my intelligence and success. 11. at times, i feel my success has been due to some kind of luck. ponekad se osjećam kao da je za moj uspjeh zaslužna neka vrsta sreće. s vremena na vrijeme, osjećam se kao da je za moj uspjeh zaslužna sreća. s vremena na vrijeme, osjećam se kao da je za moj uspjeh zaslužna neka vrsta sreće. i sometimes feel that my success is due to some sort of luck. 12. i’m disappointed at times in my present accomplishments and think i should have accomplished much more. ponekad sam razočaran/a trenutnim uspjesima i mislim da sam trebao/la mnogo više. ponekad sam razočaran/a svojim trenutnim postignućima i mislim da sam trebao/la postići puno više. ponekad sam razočaran/a svojim trenutnim postignućima i mislim da sam trebao/la postići puno više. i am sometimes disappointed at the level of my success and feel that i should of done better. 13. sometimes i’m afraid others will discover how much ponekad se bojim da će drugi otkriti koliko znanja i ponekad strahujem od toga da će drugi otkriti da mi ponekad se bojim da će drugi otkriti koliko mi znanja ili sometimes i fear that others will discover how much seemedj 2021, vol 5, no. 1 croatian cips 151 southeastern european medical journal, 2021; 5(1) knowledge or ability i really lack. sposobnosti mi zbilja nedostaje. nedostaje znanja ili sposobnosti. sposobnosti zbilja nedostaje. knowledge and ability i'm truly lacking. 14. i’m often afraid that i may fail at a new assignment or undertaking even though i generally do well at what i attempt. ponekad se bojim da neću uspjeti u novom zadatku ili poduhvatu iako obično dobro napravim ono u što se upustim. često se bojim da ću biti neuspješan/na u novome zadatku ili pothvatu iako sam obično uspješan/na u onome što radim. često se bojim da ću biti neuspješan/na u novom zadatku ili poduhvatu iako obično dobro napravim ono u što se upustim. often, i fear that i will not succeed in a new task or endeavour even though i usually do well what i embark on. 15. when i’ve succeeded at something and received recognition for my accomplishments, i have doubts that i can keep repeating that success. kada uspijem u nečemu i za to dobijem priznanje, sumnjam u to da ću taj uspjeh moći ponoviti. kada uspijem u nečemu i drugi pohvale moja postignuća, bojim se da neću ponovno biti uspješan/na u istom ili sličnom zadatku. kada uspijem u nečemu i za to dobijem priznanje, sumnjam da ću taj uspjeh moći ponoviti. when i get recognition for an achievement, i have doubts that i will be able to repeat that success. 16. if i receive a great deal of praise and recognition for something i’ve accomplished, i tend to discount the importance of what i’ve done. ako za nešto što sam postigao/la dobijem mnogo hvale i priznanja, sklon/a sam reducirati važnost toga što sam napravio/la. ako dobijem puno pohvala i komplimenata za nešto što sam postigao/la, često zanemarim važnost onoga što sam učinio/la. ako dobijem puno pohvala i priznanja za nešto što sam postigao/la, sklon/a sam umanjiti važnost onoga što sam učinio/la. if i receive praise for something i achieved, i tend to reduce the importance of what i did. 17. i often compare my ability to those around me and think they may be more intelligent than i am. često uspoređujem svoje sposobnosti s onim ljudima oko sebe i mislim da bi oni mogli biti pametniji od mene. često uspoređujem svoje sposobnosti sa sposobnostima drugih i mislim da su inteligentniji od mene. često uspoređujem svoje sposobnosti sa sposobnostima drugih oko sebe i mislim da bi oni mogli biti pametniji od mene. i often compare my abilities with people around me and think that they might be smarter than me. 18. i often worry about not succeeding with a project or examination, even though others around me have considerable confidence that i will do well. često se brinem da neću uspjeti s nekim projektom ili ispitom, iako su drugi oko mene snažno uvjereni da ću to dobro napraviti često brinem da ću biti neuspješan/na na projektu ili ispitu, iako druge osobe vjeruju da ću biti uspješan/na. često se brinem da ću biti neuspješan/na na projektu ili ispitu, iako druge osobe oko mene snažno vjeruju da ću to dobro napraviti. i often worry about the success on a project or exam even though others around me are strongly convinced that i’ll do well. 19. if i’m going to receive a promotion or gain recognition of some kind, i hesitate to tell ako trebam dobiti unapređenje ili priznanje neke vrste, oklijevam reći drugima sve dok to nije već ostvareno. ako trebam dobiti promaknuće ili nekakvu pohvalu, drugim osobama o tome ne govorim ako trebam dobiti promaknuće ili nekakvo priznanje, oklijevam reći drugima dok to nije već ostvareno. if i need to get a promotion or recognition of some kind, i hesitate to tell others until it seemedj 2021, vol 5, no. 1 croatian cips 152 southeastern european medical journal, 2021; 5(1) others until it is an accomplished fact. dok se to napokon ne ostvari. has already been achieved. 20. i feel bad and discouraged if i’m not “the best” or at least “very special” in situations that involve achievement. osjećam se loše i obeshrabreno ako nisam „najbolji/a“ ili barem „vrlo poseban/a“ u situacijama koje se tiču postignuća. osjećam se loše i obeshrabreno ako nisam „najbolji/a“ ili barem „vrlo poseban/na“ u situacijama u kojima se očekuje nekakvo postignuće. osjećam se loše i obeshrabreno ako nisam „najbolji/a“ ili barem „vrlo poseban/na“ u situacijama u kojima se očekuje nekakvo postignuće. i feel bad and discouraged if i am not "the best" or at least "very special" in situations concerning success. the clance ip scale translated into the croatian language and scoring instructions are presented in appendix 1. discussion the final version of the scale is suitable to assess the impostor phenomenon among medical students in croatia and bosnia and herzegovina. there is extensive variability in the literature associated with the impostor phenomenon, not only regarding synonyms which describe this internal feeling of inadequacy (28). some reported inconsistencies are most likely attributed to the methodological issues and methodological quality of impostorism validation studies. for our research, we selected clance’s scale, which showed to be a highly sensitive and reliable instrument (1). validation of the cip scale is beyond the scope of this research. the sample size for a validation study will be determined when the final version of the questionnaire is administered to a large representative sample of respondents for whom the questionnaire is intended – medical, veterinary and nursing students. the covid-19 pandemic prolonged the process of obtaining ethical approvals and made the research more demanding in terms of how to distribute the questionnaire to participants. ideas and suggestions for further research include checking psychometric characteristics and validating the clance impostor phenomenon scale translated into the croatian language. the version of the cips which was translated into the croatian language represents a reliable translation ready to be used in croatia and bosnia and herzegovina. acknowledgement. the authors express special gratitude to professional translators prof. jeremy white and prof. martina batinica for forward translations, and to prof. meri odak for proofreading and correcting the croatian version of the cips. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. mak kkl, kleitman s, abbott mj. impostor phenomenon measurement scales: a systematic review. front psychol. 2019;10. 2. clance pr. the impostor phenomenon: overcoming the fear that haunts your success. peachtree pub ltd; 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31(6):863–74. 23. sakulku j, alexander j. impostor phenomenon. impos phenom jaruwan. 2011; 6:1–5. 24. brauer k, wolf a. validation of the german-language clance impostor phenomenon scale (gcips). pers individ dif [internet]. 2016;102:153–8. available from: http://dx.doi.org/10.1016/j.paid.2016.06.071 25. kolligian j, sternberg rj. perceived fraudulence in young adults: is there an “impostor syndrome”? j pers assess. 1991; 56(2):308–26. 26. leary mr, patton km, orlando ae, funk ww. the impostor phenomenon: selfperceptions, reflected appraisals, and seemedj 2021, vol 5, no. 1 croatian cips 154 southeastern european medical journal, 2021; 5(1) interpersonal strategies. j pers. 2000; 68(4):725– 56. 27. chrisman sm, pieper wa, clance pr, holland cl, glickauf-hughes c. validation of the clance impostor phenomenon scale (gcips). valid clance impos phenom scale. 1995; 65(3):456–67. 28. gibson-beverly g, schwartz jp. attachment, entitlement, imposter phenomenon. j coll couns. 2008; 11(2):119–32. 29. milić j, škrlec i, vranješ im, matić m, sertić d, heffer m. the croatian translation of the horne and östberg morningness-eveningness questionnaire with a brief review of circadian typology. southeast eur med j. 2018; 2(1):1–11. .. appendix 1. the clance ip scale translated into the croatian language and scoring instructions clance ljestvica fenomena varalice molimo zaokružite broj koji najbolje odražava koliko je za vas izjava istinita. najbolje je da zaokružite prvi odgovor koji vam padne na pamet i ne premišljate se o tome. 1. često bih uspješno napisao/la test ili izvršio/la zadatak iako sam se prije početka bojao/la da ga neću dobro napraviti. 1 (nimalo istinito) 2 (rijetko) 3 (ponekad) 4 (često) 5 (posve istinito) 2. mogu odati dojam da sam sposobniji/a nego što doista jesam. 1 (nimalo istinito) 2 (rijetko) 3 (ponekad) 4 (često) 5 (posve istinito) 3. izbjegavam procjene ako je moguće i strašim se da me drugi procjenjuju. 1 (nimalo istinito) 2 (rijetko) 3 (ponekad) 4 (često) 5 (posve istinito) 4. kada me drugi hvale za moja postignuća, bojim se da u budućnosti neću moći ispuniti njihova očekivanja. 1 (nimalo istinito) 2 (rijetko) 3 (ponekad) 4 (često) 5 (posve istinito) 5. ponekad mislim da sam postigao/la sadašnju poziciju ili sadašnji uspjeh jer sam bio/la na pravom mjestu u pravo vrijeme ili sam poznavao/la prave ljude. 1 (nimalo istinito) 2 (rijetko) 3 (ponekad) 4 (često) 5 (posve istinito) 6. strahujem od toga da će meni važni ljudi otkriti da nisam sposoban/na koliko oni misle da jesam. 1 (nimalo istinito) 2 (rijetko) 3 (ponekad) 4 (često) 5 (posve istinito) 7. sklon/a sam češće se prisjetiti događaja u kojima nisam dao/la sve od sebe nego onih kada sam dao/la sve od sebe. seemedj 2021, vol 5, no. 1 croatian cips 155 southeastern european medical journal, 2021; 5(1) 1 (nimalo istinito) 2 (rijetko) 3 (ponekad) 4 (često) 5 (posve istinito) 8. rijetko kada projekt ili zadatak napravim onoliko dobro koliko bih volio/ljela. 1 (nimalo istinito) 2 (rijetko) 3 (ponekad) 4 (često) 5 (posve istinito) 9. ponekad se osjećam ili vjerujem da je uspjeh u mom životu ili poslu posljedica nekakve pogreške. 1 (nimalo istinito) 2 (rijetko) 3 (ponekad) 4 (često) 5 (posve istinito) 10. teško prihvaćam komplimente ili pohvale na račun svoje inteligencije ili postignuća. 1 (nimalo istinito) 2 (rijetko) 3 (ponekad) 4 (često) 5 (posve istinito) 11. ponekad se osjećam kao da je za moj uspjeh zaslužna neka vrsta sreće. 1 (nimalo istinito) 2 (rijetko) 3 (ponekad) 4 (često) 5 (posve istinito) 12. ponekad sam razočaran/a svojim trenutnim postignućima i mislim da sam trebao/la postići puno više. 1 (nimalo istinito) 2 (rijetko) 3 (ponekad) 4 (često) 5 (posve istinito) 13. ponekad se bojim da će drugi otkriti koliko mi znanja ili sposobnosti doista nedostaje. 1 (nimalo istinito) 2 (rijetko) 3 (ponekad) 4 (često) 5 (posve istinito) 14. često se bojim da ću biti neuspješan/na u novom zadatku ili poduhvatu premda obično dobro napravim ono u što se upustim. 1 (nimalo istinito) 2 (rijetko) 3 (ponekad) 4 (često) 5 (posve istinito) 15. kada uspijem u nečemu i za to dobijem priznanje, sumnjam da ću taj uspjeh moći ponoviti. 1 (nimalo istinito) 2 (rijetko) 3 (ponekad) 4 (često) 5 (posve istinito) 16. ako za nešto što sam postigao/la dobijem puno pohvala i priznanja, sklon/a sam umanjiti važnost onoga što sam učinio/la. 1 (nimalo istinito) 2 (rijetko) 3 (ponekad) 4 (često) 5 (posve istinito) 17. često uspoređujem svoje sposobnosti sa sposobnostima drugih oko sebe i mislim da bi oni mogli biti pametniji od mene. 1 (nimalo istinito) 2 (rijetko) 3 (ponekad) 4 (često) 5 (posve istinito) 18. često se brinem da ću biti neuspješan/na na projektu ili ispitu, premda druge osobe oko mene snažno vjeruju da ću to dobro napraviti. seemedj 2021, vol 5, no. 1 croatian cips 156 southeastern european medical journal, 2021; 5(1) 1 (nimalo istinito) 2 (rijetko) 3 (ponekad) 4 (često) 5 (posve istinito) 19. ako trebam dobiti promaknuće ili nekakvo priznanje, oklijevam reći drugima sve dok to nije već ostvareno. 1 (nimalo istinito) 2 (rijetko) 3 (ponekad) 4 (često) 5 (posve istinito) 20. osjećam se loše i obeshrabreno ako nisam „najbolji/a“ ili barem „vrlo poseban/na“ u situacijama u kojima se očekuje nekakvo postignuće. 1 (nimalo istinito) 2 (rijetko) 3 (ponekad) 4 (često) 5 (posve istinito) ocjenjivanje testa fenomena varalice test fenomena varalice osmišljen je kako bi pomogao pojedincima utvrditi imaju li ili nemaju karakteristike fenomena varalice te, ako imaju, u kojoj mjeri. nakon rješavanja testa zbrojite brojeve odgovora na svaku izjavu. ako je ukupan zbroj 40 ili manje, ispitanik ima malo obilježja fenomena varalice; ako je rezultat između 41 i 60, ispitanik ima umjerena iskustva s fenomenom varalice; rezultat između 61 i 80 znači da se ispitanik često osjeća kao varalica; a rezultat viši od 80 znači da ispitanik često intenzivno osjeća fenomen varalice. što je rezultat viši, fenomen varalice češće i ozbiljnije utječe na život osobe. bilješka. iz the impostor phenomenon: when success makes you feel like a fake (str. 20 – 22), autorice p. r. clance, 1985., toronto: bantam books. © pauline rose clance, dr. sc., american bord of professional psychology. koristi se uz dopuštenje. zabranjeno umnožavati bez dopuštenja pauline rose clance, drpaulinerose@comcast.net, www.paulineroseclance.com abbreviations. ip impostor phenomenon cips clance impostor phenomenon scale author contribution. acquisition of data: čarapina zovko i, milić j, vucemilovic fb, jemrić n, sulić p, turudić m, vidović d, jelovica d, padjen ivanac g, ivić v, škrlec i, biloglav z. administrative, technical, or logistic support: čarapina zovko i, milić j, vucemilovic fb, jemrić n, sulić p, turudić m, vidović d, jelovica d, padjen ivanac g, ivić v, škrlec i, biloglav z. analysis and interpretation of data: čarapina zovko i, milić j, vucemilovic fb, jemrić n, sulić p, turudić m, vidović d, jelovica d, padjen ivanac g, ivić v, škrlec i, biloglav z. conception and design: čarapina zovko i, milić j, vucemilovic fb, jemrić n, sulić p, turudić m, vidović d, jelovica d, padjen ivanac g, ivić v, škrlec i, biloglav z. critical revision of the article for important intellectual content: čarapina zovko i, milić j, vucemilovic fb, jemrić n, sulić p, turudić m, vidović d, jelovica d, padjen ivanac g, ivić v, škrlec i, biloglav z. drafting of the article: čarapina zovko i, milić j, vucemilovic fb, jemrić n, sulić p, turudić m, vidović d, jelovica d, padjen ivanac g, ivić v, škrlec i, biloglav z. final approval of the article: čarapina zovko i, milić j, vucemilovic fb, jemrić n, sulić p, turudić m, vidović d, jelovica d, padjen ivanac g, ivić v, škrlec i, biloglav z. guarantor of the study: čarapina zovko i, milić j, vucemilovic fb, jemrić n, sulić p, turudić m, vidović d, jelovica d, padjen ivanac g, ivić v, škrlec i, biloglav z. provision of study materials or patients: čarapina zovko i, milić j, vucemilovic fb, jemrić n, sulić p, turudić m, vidović d, jelovica d, padjen ivanac g, ivić v, škrlec i, biloglav z. seemedj 2020, vol 4, no. 1 environmental temperature and acute aortic dissection 40 southeastern european medical journal, 2020; 4(1) original article decrease in environmental temperature may trigger the onset of acute aortic dissection 1 ivan švagelj* 1#, ivan vlahović 2#, doris ogresta 3, dražen belina 4, zdenko kovač 5, 6 1 department of pathology and cytology, general county hospital vinkovci, croatia 2 department of surgery, university hospital centre osijek, osijek, croatia 3 department of gastroenterology and hepatology, “sestre milosrdnice” university hospital center, zagreb, croatia 4 department of cardiac surgery, university hospital centre zagreb, university of zagreb, school of medicine, zagreb, croatia 5 department of pathophysiology university hospital centre zagreb, zagreb, croatia 6 university of zagreb, school of medicine, zagreb, croatia # contributed equally to this paper *corresponding author: ivan švagelj, svagelj.ivan@gmail.com received: oct 21, 2019; revised version accepted: mar 9, 2020; published: apr 27, 2020 keywords: acute aortic dissection, blood pressure, cold temperature, thermotolerance abstract aim: the most important risk factors for a stanford type a acute aortic dissection (aad) include arterial hypertension and connective tissue disorders, while numerous studies have identified meteorology factors, such as environmental temperature also play an important role. the aim of this study is to explore the relationship between environmental temperature and the frequency of aad surgically threated over a 12-year period at a croatian university hospital. methods: this is a retrospective, monocenter observational study conducted at the university hospital centre zagreb. the study includes 134 patients who were threated surgically for stanford type a aad between january 2001 and december 2012. temperature categories (low, moderate and high) were based on the calculated monthly average environmental temperature and standard deviation given from official daily environmental temperatures for the respective period. results: the results show a higher frequency of aad in days of low temperature compared to days of moderate temperature or high temperature. the frequency of days with aad was somewhat higher in moderate than high temperature category, but the difference is not statistically significant. the relative frequency of aad for low, moderate and high temperature categories were 4.55, 2.96 and 1.93, respectively. conclusion: environmental temperature drop induces stressful adaptive body response, including an additional hemodynamic load and increase in arterial blood pressure, strong enough to trigger the aad-etiopathogenesis. furthermore, our findings indicate that body response to environmental heat may differ from a response to reduced environmental temperature, due to observed small number of events in days of high temperature. (švagelj* i, vlahović i, ogresta d, belina d, kovač z. decrease in environmental temperature may trigger the onset of acute aortic dissection. seemedj 2020; 4(1); 40-48) seemedj 2020, vol 4, no. 1 environmental temperature and acute aortic dissection 41 southeastern european medical journal, 2020; 4(1) introduction among cardiovascular diseases, which are the leading cause of death in the developed countries, acute aortic dissection (aad) stands out as one of the most lethal. with an in-hospital mortality rate of 22 % for stanford type a (involving ascending aorta) and about 12 % for stanford type b (no ascending aorta involvement), aad represents an emergency case in the cardiovascular surgery (1). at a histological level, aad is the tearing of the inner surface of the aortic layer known as intima, whereupon blood at high pressure splits or dissects the media to form a false channel or lumen that runs alongside the true lumen. a further re-entrance tear allows blood to circulate through the false lumen (2, 3). retrograde spreading of this tear results in either the penetration of blood into the pericardium, causing hemopericardium and heart tamponade, or the rupturing of the aortic wall. both of these scenarios are medical emergencies, as they lead to the rapid death of the patient if timely surgical intervention is not performed (2, 4). aad affects men at about twice the rate that it does women and it commonly occurs between 60 and 80 years of age (5). besides age, other well-known risk factors are an arterial hypertension, an aortic aneurism, congenital diseases of connective tissue like marfan syndrome or elhers-danlos syndrome, various types of vasculitis and a bicuspid aortic valve (6). furthermore, a number of studies have revealed meteorology factors (e.g., environmental temperature) as potentially risk factors which increase the incidence and mortality of aad during the cold months of the year (7-11). a number of published studies have compared aad with the exact data on environmental temperature and interestingly, although all the studies were carried out in a moderate zone of western europe, the results of the studies are contradictory. in fact, one group of authors from the united kingdom (12) showed that climate factors have no impact on the occurrence of aad, whereas a group of authors from germany failed to identify any relationship between air temperature and the incidence of aad. however, the german authors did point out possible association which tended to be significant, explaining it as result of a small number of events (13). on another note, a group of authors from france (9) has reported that aad has a higher incidence during colder months of year, and a research group in the netherlands (14) noted that a higher incidence of aad correlates to a lower air temperature (low minimal daily temperature). a second group from germany (15) noted that changes in the air temperature and amount of cloudiness are the most representative weather predictors among the studied parameters. the aim of this study is to explore the relationship between relative environmental temperature and the frequency of stanford type a acute aortic dissection. materials and methods this is a retrospective, monocenter observational study conducted in one university hospital located in zagreb (croatia). the study has been approved by the ethical committee of university hospital centre zagreb. information from hospital records were combined with data from local weather forecast archives. this approach assumes that all person in a specified geographic area experienced the same exposure conditions. clinical data the medical records of the patients consecutively admitted to the department of cardiac surgery at the university hospital centre zagreb in the period between 1 january 2001 and 31 december 2012 were investigated. inclusion criteria were diagnosis and surgically treatment of the patient with a stanford type a aad. the baseline characteristics of patients (gender and age), diagnosis of arterial hypertension and/or connective tissue disorder, as well as precise date and time of the first aad symptoms were recorded for each candidate. after searching operating protocols 212 patients were identified as fitting the given criteria, but 35 seemedj 2020, vol 4, no. 1 environmental temperature and acute aortic dissection 42 southeastern european medical journal, 2020; 4(1) were excluded due to onset of iatrogenic aad (2 patients) and incomplete or unclear data (33 patients). upon consultation with croatian meteorological and hydrological service (cmhs), the geographic area of the specific climate type was determined, meaning a cfb climate type according to the köppen classification. it is a temperate oceanic climate (c) without dry season (f) but with warm summers (b) (16). the geographic area of interest is a circular area with a diameter of 195 km (with zagreb as the center) and includes nine croatian counties inhabited by approximately 2.1 million inhabitants. accordingly, another 43 of the remaining 177 patients were excluded given that they reside outside of the determined geographic area at the moment of onset of aad. hence, a total of 134 patients with surgically treated stanford type a aad were included in the study. meteorological data exact data on environmental temperatures for the determined area and timeframe were collected from the official weather database provided by cmhs. all average daily temperatures of twelve-years studied period were categorized in one of the three relative temperature categories: low, moderate or high. relative temperature categories, for each month separately, were determined from the calculated average monthly temperature and standard deviation of these months (e.g. all januaries, februaries etc.) during the studied period. hence, the moderate category of each month is defined as the interval between the value of average monthly temperature plus and value of average monthly temperature minus one standard deviation. at the same way, the high and low categories of each month are defined as one standard deviation higher or lower than the average monthly temperature of these months. consequently, daily average temperatures of the studied twelve-year period which were between the monthly average temperature and one standard deviation interval in which the average daily temperature belongs to, were placed in the moderate category. daily average temperatures higher than the average monthly temperature plus one standard deviation for the month they belong to were placed in the high category, whereas average daily temperatures lower than the average monthly temperature minus one standard deviation were placed into low category (figure 1). figure 1. categorization of average daily temperatures of the studied twelve-year period in respect to the average monthly temperature and standard deviation of each month the figure shows average monthly temperature (black dots) with one standard deviation interval for each month. average monthly temperatures plus the respective standard deviation are linked together with a red line and all average monthly temperatures minus the respective standard deviation are linked together with a blue line in order to get the boundaries of relative temperature categories. all average daily temperatures which were fallen above the red line are categorized as a high temperature and those below the blue line as a low temperature. average daily temperatures which were within one standard deviation interval are categorized as a moderate temperatures. furthermore, temperature categories were divided into two groups, one for days when aad occurred and the other without aad occurrence. finally, the same categories from all months were summarized in order to obtain the final table for analysis. seemedj 2020, vol 4, no. 1 environmental temperature and acute aortic dissection 43 southeastern european medical journal, 2020; 4(1) statistical analysis descriptive statistics and statistical analysis were performed using statistical software program r (17). the normality distribution of average monthly temperatures for each month was tested using the kolmogorov-smirnov test. differences in the frequency of days with and without occurrence of aad between temperature categories was tested using the chi-square test. p-values < 0.05 were considered to be statistically significant. results according to the clinical and geographic criteria given in this study (see the materials and methods section), a total of 134 patients who were threated surgically for stanford type a aad at the department of cardiac surgery, university hospital center zagreb, between january 2001 and december 2012, were included in the data analysis. a descriptive analysis by gender shows that 89 (66.4%) of cases were men and 45 (33.6%) were women. the average age of the patients was 57.8 years with a standard deviation (sd) of 12.8, ranging from 18 to 82 years. arterial hypertension was noticed in 91 (67.9 %) patients while 25 of them (18.7 %) did not have arterial hypertension, whereas data were missing for 18 patients (13.4%). furthermore, the marfan syndrome, one of the most common connective tissue disorder, was detected in one case (0.7 %) (table 1).. table 1. patient characteristics (sd – standard deviation) patients (n = 134) gender female 45 (33.6 %) male 89 (66.4 %) age (years) youngest 18 oldest 82 mean/sd 57.8/12.8 hypertension1 with 91 (67.9 %) without 25 (18.7 %) connective tissue disorder with2 1 (0.7 %) without 133 (99.3 %) 1missing values for 18 patients; 2 marfan syndrome the difference in the frequencies of aad between the defined relative temperature categories was statistically significant (χ² = 8.65, df = 2, p = 0.01). further analysis shows a higher frequency of aad in low temperature days compared to moderate temperature days (χ² = 4.12, df = 1, p-value < 0.05) or high temperature days (χ² = 7.12, df = 1, p < 0.01) (table 2).. seemedj 2020, vol 4, no. 1 environmental temperature and acute aortic dissection 44 southeastern european medical journal, 2020; 4(1) table 2. relative temperature categories including frequencies of days (n) with and without occurrence of aad (n (%)) the difference in the frequencies of days with and without aad between the defined relative temperature categories was statistically significant (χ² = 8.65, df = 2, p = 0.01). there is a higher frequency of aad on low temperature days compared to moderate temperature days (χ² = 4.12, df = 1, p-value < 0.05*) or high temperature days (χ² = 7.12, df = 1, p < 0.01**), while the difference between moderate and high temperature categories is not statistically signifi cant (χ ² = 1.95, df = 1, p = 0.16) although the frequency of days with aad is somewhat higher in the moderate than high temperature category, this difference is not statistically significant (χ² = 1.95, df = 1, p = 0.16). to get a more comparable and evident relationship, the given results were transformed into a relative frequency of aad for each temperature category using a particular method. the number of days with aad in each temperature category were divided by all days (with and without aad) of the same category and then multiplied by 100. hence, the relative frequency of aad for low, moderate and high categories were 4.55, 2.96 and 1.93, respectively (figure 2).. figure 2. relative frequency of aad occurrence between temperature categories there is a higher relative frequency of aad occurrence for the low temperature category (4.55) compared to the moderate (2.96) and high (1.93) temperature category, while difference in frequency of aad between the moderate and high temperature is not statistically significant. the specified p-values between categories correspond with those calculated from table 2. discussion this study reports a connection between variation of environmental temperature and occurrence of aad. more than 60 percent of patients included in the present study were men, which correlates to the relevant literature (1, 10). the average age of patients is 58 years with the number of patients aged 70-79 decreasing, which deviates from previously mentioned literature (5). this may be attributed to limitations of the methodology used in the study, meaning patients who died were not included, given that had not undergone surgery and consequently had not been recorded in the surgical operation protocol. low moderate high total days with aad 33 (0,75) 87 (2) 14 (0,32) 134 (3,1) days without aad 692 (15,8) 2846 (64,9) 711 (16,23) 4249 (96,9) total 725 (16,55) 2933 (66,9) 725 (16,55) 4383 (100) seemedj 2020, vol 4, no. 1 environmental temperature and acute aortic dissection 45 southeastern european medical journal, 2020; 4(1) a human physiological, and possible pathological, outcome is strongly connected to numerous internal and external factors and their interplay which is a well-known fact. hence, arterial hypertension, as one of the most common cardiovascular diseases and a predisposing factor for blood vessel rupture, was also noted within the study and which indicates a more frequent occurrence of aad in patients with arterial hypertension (68 %), which correlates to the data in the literature (1, 18, 19). the significance of high arterial pressure in the onset of aad may be attributed to its contribution to damaging the aortic inner layer (intima) which may be the first step in the onset of aad (2, 3, 20). due to laplace’s law, a sudden increase in arterial pressure inside the aortic lumen leads to an increase in aortic wall tension, consequently increasing the risk of aortic wall rupture (6). environmental temperature is an important, but not always perceptible external factor affecting arterial pressure. hintsala et al. showed that short-term cold exposure increases central aortic blood pressure and cardiac overload, which may contribute to the observed increased winter mortality (21). a similar result is that outdoor temperature and blood pressure are strongly correlated whereby systolic blood pressure decreases with increasing temperature, but only in the elderly population (those over than 80 years of age) as reported by alperovitch et al. (22). to reduce the well-known influence of a climate type on environmental temperatures, we included 134 patients from only a single climate type (the cfb type according to the köppen classification). there has been only one study from france with a similar approach (23). our results show a higher frequency of aad for low temperatures regardless of the season and the month(s) of year. on another note, groups of authors from uk (7), former serbia and montenegro (8), korea (10) and japan (11) have reported that the frequency of aad is significantly higher in winter and peaking in january. similar results indicate that cold weather is correlates to a higher incidence of aad as reported by verberkmoes et al. from the netherlands (14). moreover, li et al. reported that the onset of stanford type b aad was higher in winter than in summer and autumn, and that a low maximal daily temperature is associated with occurrence of the stanford type b aad (24), while xie et al. noted that cold atmospheric temperature and larger daily temperature changes correlated to a higher incidence of aad (25). both of these studies were from china. in our opinion, a relative drop in environmental temperature might be more important risk factor than absolute temperature value, given the highest frequency of aad when the average daily temperature was categorized as low. this hypothesis was first mentioned in 2005, when mehta et al. reported that the winter peak for aad was evident in both cold and temperate climate settings, suggesting that the relative change in temperature, rather than absolute temperature, may be a mechanistic factor (26). benouaich et al. showed that the incidence of aortic dissection was higher in a winter time than in summer, but also that the days with occurrences of aad were colder than those without aad, concluding that a relative change in temperature may be a triggering factor in onset of aad (9). the results conform with a study by a group of authors from nantes (france) who arrived to the same conclusion, but using different methodology. they reported that, regardless of the season, a decrease in average daily temperature of more than 5°c, between days 0-7, significantly increases the risk of acute aortic syndrome at day 0 (23). finally, a group from china which tried to predict aad occurrence in respect to environmental temperature changes, concluded that for every 10 °c increase, the incidence drops by 0.21 units (27). an explanation of the possible pathophysiologic mechanism for aad occurrence regarding temperature changes may be activation of body temperature regulators such as thermogenesis, cytokine response, hemodynamic adaptation and hypermetabolism (6). the human organism retains the heat by decreasing the release of heat (i.e. vasoconstriction) and increasing heat generation (i.e. increased metabolism) what attribute to sympathetic activity increase. (6, 9, 14, 21). consequently, vasoconstriction and an increased metabolism (including a higher seemedj 2020, vol 4, no. 1 environmental temperature and acute aortic dissection 46 southeastern european medical journal, 2020; 4(1) cardiac work load) increases mean arterial pressure, which is inversely proportional to changes in body temperature, and in fact environmental temperature (6, 9, 14, 21). edwin et al. reported that the pathogenesis of aad may result from the interaction of three factors: 1) an existing pathological condition of the aortic media, 2) any agent of intimal injury or tear, and 3) hemodynamic factors that propagate the dissection once it has been initiated (28). hence, a possible explanation of the connection between a drop in environmental temperature and aad occurrence is attributed to an increase in arterial pressure leading to a critical level when intimal injury occurs and consequently propagation to aad caused by the same factor. this value of critical level of arterial pressure is unique for each person individually, while increasing in arterial pressure is generated by the activation of above-mentioned defense (sympathetic) mechanisms. the assumption is that a drop in environmental temperature induces a stressful, adaptive response from the body, including an additional hemodynamic load (and consequently increasing arterial pressure), which is strong enough to trigger and propagate the aad etiopathogenesis. also, our findings indicate that body response to an environmental heat may differ from the response to reduction in environmental temperature, given the big difference in aad occurrence between low and high temperature categories. these results suggest that a change in environmental temperature does not necessarily lead to a higher frequency of aad. instead the temperature must decrease. moreover, the increase in environmental temperature may have a “protective” effect, given that the lowest frequency of aad occurs for high temperatures what conforms with results reported by law et al. (27). however, the difference between the moderate and high temperature category is not significant, and it may be due to the fact that the vast majority of average daily environmental temperatures during the year fall into the category of moderate temperatures (~66%) and as a consequence, aad as a multifactorial disease, occurs just below these conditions. consequently, this relationship may be confirmed using a larger number of events. another limitation of the study should be pointed out. the university hospital centre zagreb is not the only institution where aad is surgically threated, hence an unknown number of events in the investigated area and period were not included in this study. conclusion present qualitative retrospective study highlights a dependence between changing of environmental temperatures and aad occurrence, thus indicating a possible causative correlation, what provides the basis for further thesis research in a future multicenter study. acknowledgement. the article is a part of the study which was c award in 2013 at the university of zagreb croatia under the mentorship of cardiovascular surgeon dražen belina and university professor zdenko kovač. special acknowledgement to croatian meteorological and hydrological service for providing of weather data. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. evangelista a, maldonado g, gruosso d, teixido g, rodríguez-palomares j, eagle k. insights from the international registry of acute aortic dissection. glob cardiol sci pract. 2016; 2016(1):e201608. doi: 10.21542/gcsp.2016.8. 2. ahmad f, cheshire n, hamady m. acute aortic syndrome: pathology and therapeutic strategies. postgrad med j 2006; 82(967):305312. 3. coady ma, rizzo ja, elefteriades ja. pathologic variants of thoracic aortic dissections: penetrating atherosclerotic ulcers and seemedj 2020, vol 4, no. 1 stigmatization of patients with viral infections and mass psychogenic illness 47 southeastern european medical journal, 2020; 4(1) intramural hematomas. cardiol clin 1999; 17(4):637-57. 4. mussa ff, horton jd, moridzadeh r, nicholson j, trimarchi s, eagle ka 2016. acute aortic dissection and intramural hematoma: a systematic review. jama 2016; 316(7):754-763. 5. damjanov i, jukić s, nola m patologija. zagreb: medicinska naklada, 2014. 6. gamulin s, marušić m, kovač z patofiziologija. zagreb: medicinska naklada, 2018. 7. mehta rh, manfredini r, hassan f, sechten u, bossone e, oh jk, cooper jv, smith de, portaluppi f, penn m, hutchison s, nienaber ca, isselbachter em, eagle ka. chronobiological patterns of acute aortic dissection. circulation 2002; 106:1110-1115. 8. lasica rm, perunicic j, mrdovic i, vujisic tesic b, stojanovic r, milic n, simic d, vasiljevic z. temporal variations at the onset of spontaneus acute aortic dissection. int heart j 2006; 47:585-595. 9. benouaich v, soler p, gourraud pa, lopez s, rousseau h, marcheix b. impact of meteorological conditions on the occurrence of acute type a aortic dissection. interact cardiovasc thorac surg 2009; 10:403-407. 10. ryu hm, lee jh, kwon ys, park sh, lee sh, bae mh, lee jh, yang dh, park hs, cho y, chae sc, jun je, park wh. examining the relationship between triggering activities and the circadian distribution of acute aortic dissection. korean circ j 2010; 40:565-572. 11. sumiyoshi m, kojima s, arima m, suwa s, nakazato y, sakurai h, kanoh t, nakata y, daida h. circadian, weekly, and seasonal variation at the onset of acute aortic dissection. am j cardiol 2002; 89(5):619-23. 12. repanos c, chadha nk. is there a relationship between weather conditions and aortic dissection? bmc surg 2005; 5.1: 21. 13. majd p, madershahian n, sabashnikov a, weber c, ahmad w, weymann a, heinen s, merkle j, eghbalzadeh k, wippermann j, brunkwall j, wahlers t. impact of meteorological conditions on the incidence of acute aortic dissection. ther adv cardiovasc dis. 2018; 12(12):321-326. 14. verberkmoes nj, soliman hamad ma, ter worst jf, tan mesh, peels ch, van straten ahm. impact of temperature and atmospheric pressure on the incidence of major acute cardiovascular events. neth heart j 2012; 20:193196. 15. taheri shahraiyni h, sodoudi s, cubasch u. weather conditions and their effect on the increase of the risk of type a acute aortic dissection onset in berlin. int j biometeorol 2016; 60: 1303. 16. šegota, t, filipčić a. köppen's classification of climates and the problem of corresponding croatian terminology. geoadria 2003; 8(1):17-37. 17. r core team. 2019 r: a language and environment for statistical computing. r foundation for statistical computing, vienna, austria. url https://www.r-project.org/. 18. nienaber ca, eagle ka. aortic dissection: new frontiers in diagnosis and management: part i: from etiology to diagnostic strategies. circulation 2003; 108:628-635. 19. klompas m. does this patient have an acute thoracic aortic dissection? jama 2002; 287(17):2262-72. 20. macura kj, corl fm, fishman ek, bluemke da. pathogenesis in acute aortic syndromes: aortic dissection, intramural hematoma, and penetrating atherosclerotic aortic ulcer. am j roentgenol 2003; 181(2):309316. 21. hintsala h, kandelberg a, herzig kh, rintamäki h, mäntysaari m, rantala a, antikainen r, keinänen-kiukaanniemi s, jaakkola jj, ikäheimo tm. central aortic blood pressure of hypertensive men during short-term cold exposure. am j hypertens 2014; 27(5):656-664. 22. alpérovitch a, lacombe jm, hanon o, dartigues jf, ritchie k, ducimetière p, tzourio c. relationship between blood pressure and seemedj 2020, vol 4, no. 1 stigmatization of patients with viral infections and mass psychogenic illness 48 southeastern european medical journal, 2020; 4(1) outdoor temperature in a large sample of elderly individuals: the three-city study. arch int med 2009; 169(1):75-80. 23. guillaume g, simon n, antoine m, sobocinski j, sénage t, pascal d, gourraud pa, blandine m. impact of relative change in temperature and atmospheric pressure on acute aortic syndrome occurrence in france. sci rep 10(1):76 doi: 10.1038/s41598-019-56841w. 24. li y, ji c, zhang j, han y. the effect of ambient temperature on the onset of acute stanford type b aortic dissection. vasa 2016; 45(395), e401. 25. xie n, zou l, ye l. the effect of meteorological conditions and air pollution on the occurrence of type a and b acute aortic dissections. int j biometeorol. 2018; 62(9):16071613. doi: 10.1007/s00484-018-1560-0. 26. mehta rh, manfredini r, bossone e, hutchison s, evangelista a, boari b, cooper jv, smith de, o'gara pt, gilon d, pape la, nienaber ca, isselbacher em, eagle ka. does circadian and seasonal variation in occurrence of acute aortic dissection influence in‐hospital outcomes? chronobiol int 2005; 22(2):343-51. 27. law y, chan yc, cheng sw. influence of meteorological factors on acute aortic events in a subtropical territory. asian j surg 2017; 40.5: 329-337. 28. edwin f, aniteye ea, sereboe l, frimpongboateng k. ecomment: acute aortic dissection in the young–distinguishing precipitating from predisposing factors. interact cardiovasc thorac surg 2009; 9:368. doi: 10.1510/icvts.2009.202234b. seemedj 2021, vol 5, no. 2 chronic kidney disease related anemia 44 southeastern european medical journal, 2021; 5(2) review article chronic kidney disease related anemia a narrative review 1 martina hrvačić *1, mariana penava 2, ana juras 3 1 university hospital “sveti duh”, clinical pharmacology unit, laboratory of clinical pharmacology, zagreb, croatia 2 university psychiatric hospital vrapče, biochemical-hematological laboratory, zagreb, croatia 3 university hospital centre zagreb, department of laboratory diagnostics, department of cytogenetics, zagreb, croatia *corresponding author: martina hrvačić, martina.hrvacic@gmail.com received: jul 31, 2021; revised version accepted: nov 4, 2021; published: nov 26, 2021 keywords: chronic kidney disease, anemia, iron abstract iron is one of the most important essential elements, required by every cell in the body. anemia is one of the most common medical conditions, defined as a decrease in blood's ability to transport oxygen to tissues, resulting in tissue hypoxia. however, anemia is not a disease, but rather the manifestation of an underlying disorder or disease and it is an important clinical marker of a disorder that may be basic or something more complex. therefore, once anemia has been diagnosed, the physician must determine its exact cause. anemia is frequently associated with chronic kidney disease (ckd) as a consequential disorder of iron metabolism and erythropoiesis regulation. it is a result of a relative erythropoietin deficiency, functional iron deficiency, impaired iron absorption, or blood loss due to dialysis. ckd related anemia is associated with an increased risk of morbidity and mortality. given the importance of public health, it is necessary to raise ckd awareness, and to encourage early diagnosis and treatment. the recommendations and guidelines of all professional societies of nephrology emphasize early diagnosis and timely treatment. it is important to ensure that patients have a good quality of life while also minimizing the risks of further complications associated with anemia. (hrvačić m, penava m, juras a. chronic kidney disease related anemiaa narrative review. seemedj 2021; 5(2); 44-57) seemedj 2021, vol 5, no. 2 chronic kidney disease related anemia 45 southeastern european medical journal, 2021; 5(2) introduction iron is involved in many cellular processes and is one of the most important minerals in the body. its absorption, transfer and metabolism are strictly regulated. most of the ironin the human body, about 60-70%, is embedded into the erythrocyte hemoglobin, while ferritin and hemosiderin account for 20-30% of iron reserves in hepatocytes and macrophages of the reticuloendothelial system (1). minimal content of iron is found in myoglobin or is embedded into various enzymes. transferrin is a transport protein in plasma and approximately 3 mg of iron is bound to it. the body of an adult contains 3-5 g of iron, with the most of the circulating iron, about 18-20 mg per day, spent on the synthesis of hemoglobin in erythroblasts.this article is written as a narrative review that provides synthesis of knowledge and applicability of results of recent and significant studies to practice. disorders of iron homeostasis anemia is one of the most common medical conditions, because it is a symptom of another disease in more than 50% of the cases. the most common causes of anemia are: decreased production, increased deterioration or loss of erythrocytes. decreased erythrocyte production is caused by weakened bone marrow, either due to a lack of bone tissue or due to suppressed inflammatory or tumor cells. erythropoietin is a hormone that stimulates the production of erythrocytes and its low levels may result inanemia (2). the main causes of such anemia are decreased production and excretion of erythropoietin (epo) from the kidneys and a reduced erythropoietic response to epo (2). what causes anemia development in ckd and what consequences does it bring? ckd is a long-term condition with progressive impairment of renal function. symptoms develop in stages, and they can generally include fatigue, nausea, vomiting, stomatitis, anorexia, muscle twitching, cramps, peripheral neuropathy, convulsions, and itching. anemia occurs in addition to this wide range of symptoms. ckd develops as a result of any condition that causes functional kidneyimpairment of sufficient duration and intensity; thus the cause of its development can be diabetic nephropathy, hypertensive nephroangiosclerosis, primary and secondary glomerulopathiesinterstitial disease, etc. metabolic syndrome associated with arterial hypertension and type 2 diabetes is a major cause of renal dysfunction (3). ckd involves five stages of kidney damage. in stage 1 ckd, egfr > 90 ml/min/1.73 m2 signifies normal glomerular filtration, but there are other signs of kidney damage, such as proteinuria or erythrocyturia. in stage 2, the egfr is between 60 and 89 ml/min/1.73 m2, and stage 3 includes 3.a.) a stagein which the egfr is between 45 and 59 ml/min/1.73 m2, and 3.b.) a stage in which the egfr is between 30 and 44 ml/min/1.73 m2, when severe symptoms appear, e.g., swelling of extremities, back pain, high blood pressure and anemia. in stage4 of the disease, the egfr is between 15 and 29 ml/min/1.73 m2, which indicates severe kidney damage in addition to the previously described symptoms. stage 5 ckd indicates end-stage renal failure and egfr is <15 ml/min/1.73 m2 (4). symptoms ofrenal failure are itching, nausea, vomiting, swelling of the extremities, pain and problems urinating, breathing and sleeping. in this stage of the disease, the patient is forced to undergo kidney dialysis or, if possible, a kidney transplant. normocytic normochromic anemia occurs in the early stages of ckd, andit is an important factor in the development of cardiovascular disease. the cause of anemia is erythropoietin deficiency. as renal tissue function is slowly lost, erythropoietin secretion decreases, and advanced renal impairment contributes to the severity of anemia. symptoms of anemia appear when gf drops below 60 ml/min in men and below 50 ml/min in women. acute anemiaspresent more severe symptoms than chronic anemias, because there is less time for the organism to adapt. significant symptoms, such as shortness of breath, seemedj 2021, vol 5, no. 2 chronic kidney disease related anemia 46 southeastern european medical journal, 2021; 5(2) weakness, fatigue, headache, loss of concentration orpalpitations, occur when hemoglobin is less than 90 g/l. iron accumulation disorders, which represent a heterogeneous group of hereditary and acquired disorders, are at the opposite end of the spectrum of iron metabolism disorders. iron overload can lead to the formation of reactive oxygen species (ros), which can damage many cellular components (5). impairediron recycling, mediated by the liver hormone hepcidin, leads to the second most common anemia: inflammatory anemia (ia) or chronic disease anemia (cda). inflammatory anemia (ia, formerly referred to as chronic disease anemia or chronic disorder anemia) is usually a mild to moderate anemia (hemoglobin rarely below 8 g/dl) that develops as a result of an infection, inflammatory disease or cancer. it differs from iron deficiency anemia in that iron stores are preserved in the marrow of macrophages, in the spleen and in the liver, which recycle obsolete erythrocytes. accordingly, inflammation anemia signifies a disorder of iron distribution. ia includes low serum iron levels despite adequate systemic iron stores (6). there are three mechanisms of inflammatory anemia, which include: a slight shortening of erythrocyte lifespan, a decrease in erythropoiesis due to a decrease in epo secretion, a poorer bone marrow response to erythropoietin and impaired cellular iron metabolism. anemia in ckd patients ckd diagnosisis based on laboratory indicators of renal dysfunction, imaging methods and renal biopsy. anemia is very common in patients with ckd and is the result of dysregulation of iron metabolism and erythropoiesis. it occurs as the result of relative erythropoietin deficiency, functional iron deficiency, impaired iron absorption, or blood loss due to dialysis. chronic renal patients with anemia have increased levels of serum hepcidin-25. patients with ckd suffer from both absolute and functional iron deficiency. absolute iron deficiency is defined by severely reduced or absent iron stores, while functional iron deficiency is defined by adequate iron stores but insufficient iron availability for incorporation into erythroid precursors, as a result of elevated hepcidin levels. absolute iron deficiency is defined when transferrin saturation (tsat) is ≤20% and serum ferritin concentration is ≤100 ng/ml among dialysis and peritoneal dialysis patients or ≤200 ng/ml among hemodialysis patients. functional iron deficiency is characterized by tsat ≤20% and elevated ferritin levels. treatment the treatment of anemia in patients with ckd is based on guidelines. the kdigo (kidney disease: improving global outcomes) group has published treatment guidelines for anemia in chronic renal patients, and the erbp (european renal best practice) group has reviewed those guidelines. the croatian society for nephrology, dialysis and transplantation (hdndt) has published its own guidelines based on the recommendations and experiences of european and international professional societies, as well as its own. in all cases of ckd or anemia, the following needs to be checked: erythrocyte count (e), hemoglobin concentration (hb), erythrocyte mean corpuscular volume (mcv), mean corpuscular hemoglobin (mch),mean corpuscular hemoglobin concentration (mchc), leukocyte count (l), platelet count (plt), absolute reticulocyte count (rtc), serum ferritin concentration, plasma transferrin saturation (tsat) and levels of vitamin b12, and folic acid (7).esa should be used with caution by people who have a history of cancer or stroke. these conditions are not absolute contraindications to the use of esa, and the decision should be made with the patient’s consent, taking the risks and benefits of the treatmentinto account. it is recommended that hb levels are maintained between 100 and 120 g/lin adults and between 110 and 120 g/lin children (7). formula for calculating iron requirements: iron (mg) = body mass (kg) x desired hb concentration (g/l) current hb concentration (g/l) x 0.24 +amount to replenish the iron stores seemedj 2021, vol 5, no. 2 chronic kidney disease related anemia 47 southeastern european medical journal, 2021; 5(2) (adults 500 mg, children 15 mg/kg of body mass) (7). table 1. main causes of ckd cause example high blood pressure (hypertension) and diabetes malignant glomerulosclerosis nephroangiosclerosis glomerulopathies primary focal glomerulosclerosis idiopathic crescentic gn iga nephropathy membranoproliferative gn membranous nephropathy with systemic diseases amyloidosis diabetes hus postinfectious gn sle wegener's granulomatosis chronic tubulointerstitial nephritis hereditary nephropathies alport's syndrome medullary cystic disease nail-chip syndrome polycystic nephropathy urinary tract obstructions by kidney stones, enlarged prostate or cancer bph urethral obstruction (congenital, stones, malignancy) vesicourethral reflux renal macrovascularopathy (artery and vein) nephrotic syndrome recurrent kidney infection (pyelonephritis) lupus and other immune system diseases including polyarteritis nodosa, sarcoidosis, goodpasture syndrome and henoch-schonlein purpura table 1. lists the main causes of ckd (8) https://my.clevelandclinic.org/health/articles/hypertension-high-blood-pressure https://my.clevelandclinic.org/health/articles/diabetes-basics https://my.clevelandclinic.org/health/articles/5989-nephrotic-syndrome https://my.clevelandclinic.org/health/diseases/15456-kidney-infection-pyelonephritis https://my.clevelandclinic.org/health/diseases/4875-lupus seemedj 2021, vol 5, no. 2 chronic kidney disease related anemia 48 southeastern european medical journal, 2021; 5(2) filtered erythrocytes should be used as needed. blood transfusion should be used in case of inadequate response to esa treatment and in cancer patients at risk for esa treatment, in case the risk outweighs its benefit. it is not recommended to use blood transfusions based on hb levels, but only in the presence of symptomatic anemia, and it is recommended in acute conditions in cases of bleeding, unstable angina pectoris and in perioperative patient care. due to the very narrow therapeutic range in which the hb concentration should be maintained and large individual differences between the patients and the methods of renal function replacement, it is very difficult to maintain the hb concentration within the set limits. epoetin therapy the introduction of erythropoietin (epo) into clinical practicefundamentallyaltered the care of patients with ckd.the extensive use of epo and its analogues (erythropoietin-stimulating agents, esas) for the purpose of anemia correction has resulted in reducing associated morbidity and improving functionality, exercise tolerance, cognitive function, and overall quality of life(9).patients who develop pure isolated red cell aplasia (prca) after treatment with any erythropoietin should not receive epoetin alfa (10). increased incidence of thrombotic vascular events (tvd) has been observed in patients receiving drugs to stimulate erythropoiesis. these include venous and arterial thrombosis and embolism (including some fatalities), such as deep vein thrombosis, pulmonary embolism, retinal vein thrombosis, and myocardial infarction. in addition, cerebrovascular events have been reported (including stroke, cerebral hemorrhage, and transient ischemic attacks) (11). darbepoetin alfa darbepoetin alfa is a second-generationesa that is a supersialylated analogue of epo, possessing two extra n-linked glycosylation chains. this property confers a lower clearance rate in vivo, and the elimination half-life of the compound in humans after a single intravenous administration is 25.3 hours versus 8.5 hours for epoetin alfa. it has a 3-fold longer serum half-life compared to epoetin alpha and epoetin beta. it stimulates erythropoiesis (increases red blood cell levels) by the same mechanism as rhuepo (binding and activating the epo receptor) and is used to treat anemia commonly associated with ckd and cancer chemotherapy (12). methoxy polyethylene glycol – epoetin beta (cera) alternative bioengineering techniques for extending the half-life of epo resulted in the development of cera, a continuous erythropoietin receptor activator. the drug stimulates erythropoiesis by interacting with the erythropoietin receptor on progenitor cells in the bone marrow.cera has an elimination half-life in humans that is considerably longer than the half-life of either epoetin or darbepoetin alfa (13).stage 4 studies suggest that many patients are able to be maintained with monthly administration of cera, and a superiority study suggests greater efficacy with this frequency of administration compared with monthly dosing of darbepoetin alfa when administered intravenously to hemodialysis patients. effects of therapy in ckd in ckd, other causes of anemia must be corrected before initiating epoetin therapy. if the serum ferritin concentration falls below 100 ng/ml, it is necessary to introduce iron therapy. it is best to give intravenous iron, although oral administration can also be considered in patients not yet on dialysis. in time to reconsider evidence for anaemia treatment (treat's) largest essential safety arguments (esa) study, initiating esa therapy in patients with mild anemia underwent a benefit – risk assessment and was found to be unfavorable due to a small increase in quality of life, and high possibility of stroke (14). based on research, the kidney disease: improving global outcomes (kdigo) guidelines recommend the introduction of esa therapy when the hemoglobin concentration falls below 9 g/dl. the commonly used target range of hemoglobin is seemedj 2021, vol 5, no. 2 chronic kidney disease related anemia 49 southeastern european medical journal, 2021; 5(2) between 10 and 12 g/dl. the usual initial intravenous or subcutaneous dose of epoetin is about 25 to 50 iu/kg two or three times a week, of darbepoetin alfa 20 to 30 mcg once a week, and of c.e.r.a. 30 to 60 mcg once every two weeks. an increase in reticulocytes (rtc) is observed 3 to 4 days after startingthe therapy, and an increase in hemoglobin of about 1 to 2 g/dlis observed during one month. it is not recommended to increase the hemoglobin level to 13 g/dl; instead, the limit should be at 11.5 g/dl (15). hyporesponsiveness to esa therapy according to the latest recommendations, hyporesponsiveness to esa therapy occurs when the hb concentration does not respond by increasing in the first month of esa treatment using modified weight-based dosages, or if, after treatment with stable doses, the patient’s esa dosage is changed two times up to 50% of the dose previously used, and that earlier dose achieved a stable condition. the usualcauses of hyporesponsiveness are iron deficiency, infection or inflammation and underdialysis. if possible, the causes of hyporesponsiveness must be treated and corrected. for example, if there is a possible iron deficiency, the patient should receive iron treatments intravenously. using laboratory tests, it is possible to establish if a patient has severe hyperparathyroidism, or a deficiency of vitamin b12, folate and thyroxine. hb electrophoresis should be performed to rule out hemoglobinopathies as causes of resistance to esa therapy. some patients taking angiotensin converting enzyme (ace) inhibitors or angiotensin receptor blockers may be expected to require a higher dose of esa treatment, but it is rarely necessary to stop taking these drugs. if a primary bone marrow disorder is suspected, a hematological examination of the bone marrow sample should be performed. a bone marrow test is also performed when antibody mediated prca are suspected, although a reticulocyte test and antibody to epo measurement can be performed prior to this test. if a patient receiving esa therapy has a high count of rtc, the bone marrow produces more new erythrocytes than necessary. bleeding or hemolysis should be investigated by endoscopic examination or hemolysis test (serum bilirubin, coomb’s test,ldh lactate dehydrogenase and haptoglobin levels). the maximum dose of the drug is not precisely defined and doses of 60,000 iu epo per week are commonly used in the united states. there are indications that high doses of the drug may increase the side effects regardless of hb concentration. experience has shown that very high doses can be effective even in critical patients, butrtc indicate no reduction in transfusion requirements and a rise in deep vein thrombosis. given all that, it seems reasonable to continue with the same dose of esa (16). pharmacotherapy using roxadustat roxadustat, a new drug approved in japan and china, is used in the treatment of anemia and it reacts as hypoxia-inducible factor prolyl hydroxylase inhibitor (hif-ph), thus increasing endogenouserythropoietin production, which stimulates the production of hemoglobin and erythrocytes. roxadustat activates a response that occurs naturally when the body responds to reduced blood oxygen levels. roxadustat promotes erythrocyte production by increasing endogenous erythropoietin production, improving the absorption, transport and utilization of iron and reducing hepcidin regulation, which helps overcome the negative impact of inflammation on hemoglobin synthesis and production of erythrocytes (17). studies a list of studies conducted in ckd patients with anemia can be found on the website https://clinicaltrials.gov/(18). a total of 337 studies were found for anemia in chronic kidney disease, five of which are currently active and are listed in table 2.a total of 243 studied were completed. table 3. shows the first 30 completed studies involving different countries of the world (18). insights provided by these medical research promises to lessen the impact of anemia in ckd patient by making sure that existing treatments are used in the best possible seemedj 2021, vol 5, no. 2 chronic kidney disease related anemia 50 southeastern european medical journal, 2021; 5(2) ways. research can find answers to things that are unknown, filling gaps in knowledge and changing the way that health care professionals work and ultimately improving ckd patient treatment and care. . table 2. list of studies conducted in chronic renal patients with anemia, clinicaltrials.gov (16) row status study title conditions interventions locations 1. active, not recruiting study of hemax pfs versus eprex/erypo® in predialysis chronic kidney disease anemia in chronic kidney disease biological: erythropoetin alfa • cemedic buenos aires, argentina • cereha buenos aires, argentina • cimel buenos aires, argentina • and 8 more… 2. active, not recruiting ascertain the optimal starting dose of mircera given subcutaneously for maintenance treatment of anemia in pediatric patients with chronic kidney disease on dialysis or not yet on dialysis anemia, renal insufficiency, chronic drug: mircera • university of alabama at birmingham; pediatric nephrology birmingham, alabama, united states • loma linda university health, loma linda, california, united states • emory university school of med; pediatrics atlanta, georgia, united states • and 19 more… 3. active, not recruiting desidustat in the treatment of anemia in ckd chronic kidney disease stage 3, anemia, chronic kidney disease stage 4, chronic kidney disease stage 5 drug: desidustat oral tablet drug: darbepoetin alfa • sunrise hospital vijayawada, andhra pradesh, india • max super specialty hospital new delhi, delhi, india • thakershey charitable trust hospital ahmadabad, gujarat, india • and 29 more 4. active, not recruiting acute effects of intravenous iron on oxidative stress and endothelial dysfunction in non-dialysis ckd renal anemia, iron toxicity, oxidative stress, endothelial dysfunction drug: sodium chloride 0.9% intravenous solution, drug: ferinject • "dr. carol davila" teaching hospital of nephrology bucharest, romania 5. active, not recruiting prospective observational study of erythropoietiniron interaction in anemia of renal disease anemia of end stage renal disease other: specimen collection • university of louisville, university kidney center louisville, kentucky, united states • western new england renal and transplant associates springfield, massachusetts, united states • duke university durham, north carolina, united states seemedj 2021, vol 5, no. 2 chronic kidney disease related anemia 51 southeastern european medical journal, 2021; 5(2) table 3. list of completed studies, showing the first 30 completed studies involving different countries of the world. row study title conditions interventions locations 1. roxadustat in the treatment of anemia in chronic kidney disease patients not requiring dialysis anemia in chronic kidney disease in non-dialysis patients drug: roxadustat drug: placebo site by37503 brest, belarus site by37504 gomel, belarus site by37501 grodno, belarus and 135 more… 2. roxadustat in the treatment of anemia in chronic kidney disease (ckd) patients, not on dialysis, in comparison to darbepoetin alfa anemia in chronic kidney disease in non-dialysis patients drug: roxadustat drug: darbepoetin alfa site at43009 vienna, austria site by37503 brest, belarus site by37501 grodno, belarus (and 122 more...) 3. asp1517 phase 2 clinical trial double-blind study of asp1517 for the treatment of anemia in chronic kidney disease patients not on dialysis anemia in chronic kidney disease patients not on dialysis drug: asp1517 drug: placebo chubu, japan hokkaido, japan kansai, japan and 4 more… 4. study of fg-4592 in subjects with chronic kidney disease in china anemia in chronic kidney disease drug: fg-4592 drug: placebo peking union medical college hospital beijing, china peking university first hospital beijing, china sichuan provincial people's hospital chengdu, china (and 10 more...) 5. effect of hemodialysis on the pk of jtz-951 in subjects with end-stage renal disease anemia in chronic kidney disease drug: jtz-951 minneapolis, minnesota, united states seemedj 2021, vol 5, no. 2 chronic kidney disease related anemia 52 southeastern european medical journal, 2021; 5(2) 6. a non-interventional study of diafer in subjects with ckd on haemodialysis for treatment of iron deficiency anemia in chronic kidney disease drug: 5% iron isomaltoside 1000 heleneholmsdialysen malmö, sweden morriston hospital, renal department swansea, wales, united kingdom 7. strategies using darbepoetin alfa to avoid transfusions in chronic kidney disease anemia in chronic kidney disease patients not on dialysis biological: darbepoetin alfa other: placebo research site anniston, alabama, united states research site birmingham, alabama, united states research site huntsville, alabama, united states and 246 more… 8. targtepo treatment for anemia in chronic kidney disease (ckd) patients and end-stage renal disease (esrd) chronic kidney disease, end-stage renal disease biological: mdgn201 targtepo barzili medical center ashkelon, israel meir medical center kfar saba, israel medical center of the galilee nahariya, israel (and 2 more...) 9. a study of fg-4592 for the treatment of anemia in chronic kidney disease patients not receiving dialysis ckd anemia drug: fg-4592 drug: placebo investigational site huntsville, alabama, united states investigational site tempe, arizona, united states investigational site alhambra, california, united states (and 142 more…) 10. safety and tolerability of fcm vs standard of care in treating iron deficiency anemia in chronic kidney disease patients anemia drug: ferric carboxymaltose drug: standard medical care (smc) luitpold pharmaceuticals norristown, pennsylvania, united states seemedj 2021, vol 5, no. 2 chronic kidney disease related anemia 53 southeastern european medical journal, 2021; 5(2) 11. comparison of darbepoetin alpha and recombinant human erythropoietin for treatment of anemia in children with chronic kidney disease anemia of chronic kidney disease drug: recombinant human erythropoietin drug: darbepoetin alfa sir ganga ram hospital new delhi, delhi, india 12. a pilot study of krx-0502 (ferric citrate, administered without food, in treating iron-deficiency anemia anemia of chronic kidney disease drug: krx-0502 barzilai medical center ashkelon, israel, western galilee hospital nahariya, israel, nazareth hospital emms nazareth, israel 13. krx-0502 (ferric citrate) for the treatment of ida in adult subjects with ndd-ckd anemia of chronic kidney disease drug: ferric citrate drug: placebo akdhc medical research services, llc phoenix, arizona, united states southwest kidney institute tempe, arizona, united states, california renal research glendale, california, united states (and 33 more…) 14. a study to evaluate efficacy and safety of jtz951 compared to darbepoetin alfa in korean renal anemia patients receiving hemodialysis. anemia of chronic kidney disease drug: jtz-951 drug: darbepoetin alfa smg-snu boramae medical center seoul, korea 15. vitamin d as a modifier of serum hepcidin in children with chronic kidney disease anemia of chronic kidney disease drug: cholecalciferol johns hopkins university baltimore, maryland, united states seemedj 2021, vol 5, no. 2 chronic kidney disease related anemia 54 southeastern european medical journal, 2021; 5(2) 16. phase 2a study to evaluate prs-080 in anemic chronic kidney disease patients anemia of chronic kidney disease biological: prs-080#022-dp biological: prs-080placebo#001 university hospital brno brno, czechia, hds klaudian's hospital, mladá boleslav, czechia, institute of clinical and experimental medicine (icem) prague, czechia (and 3 more...) 17. ckd-11101 phase 3 iv study in patients who had renal anemia receiving hemodialysis anemia of chronic kidney disease biological: ckd-11101 biological: nesp / 18. effect of erythropoiesis stimulating agent therapy in patients receiving palliative care of chronic kidney disease anemia of chronic kidney disease drug: erythropoiesis-stimulating agent prince of wales hospital, chinese university of hong kong shatin, new territories, hong kong 19. ckd-11101 phase 3 sc study anemia of chronic kidney disease biological: ckd-11101 (darbepoetin alfa) biological: nesp (darbepoetin alfa) / 20. comparison study of two iron compounds for treatment of anemia in hemodialysis patients anemia of chronic kidney disease drug: supplementation of ferric carboxymaltose drug: supplementation of iron sucrose medical university of vienna, division of nephrology and dialysis vienna, austria, wiener dialysezentrum gmbh vienna, austria 21. safety, tolerability, pk & pd study of jtz-951 in anemic subjects with end-stage renal disease anemia of chronic kidney disease drug: jtz-951 drug: placebo lakewood, colorado, united states miami, florida, united states orlando, florida, united states (and 2 more…) seemedj 2021, vol 5, no. 2 chronic kidney disease related anemia 55 southeastern european medical journal, 2021; 5(2) 22. health care personnel time for anemia management with erythropoiesis stimulating agents in hemodialysis centers in croatia renal anemia of chronic kidney disease other: no intervention bjelovar, croatia split, croatia zadar, croatia zagreb, croatia 23. a study to assess hemoglobin level depending on the comorbidity index in chronic kidney disease (ckd) participants not in dialysis treated with methoxy polyethylene glycol epoetin beta (comete) renal anemia of chronic kidney disease drug: methoxy polyethylene glycol-epoetin beta chp aix aix en provence, france ch notre dame misericorde; hemodialyse ajaccio, france chi d alencon; nephrologie hemodialyse alencon, france (and 105 more…) 24. mass balance study of jtz-951 in subjects with end-stage renal disease on hemodialysis anemia of chronic kidney disease drug: jtz-951, 14c-jtz-951 minneapolis, minnesota, united states 25. does oral pentoxifylline administration improve hemoglobin in hemodialysis patients? anemia of chronic kidney disease drug: pentoxifylline tanta university hospital tanta, egypt 26. study to evaluate effect of lapatinib on pharmacokinetics of jtz-951 in subjects with end-stage renal disease anemia of chronic kidney disease drug: jtz-951 drug: lapatinib minneapolis, minnesota, united states 27. periodic versus continuous iv iron supplementation in hd patients anemia of chronic kidney disease drug: iron sucrose supplement papageorgiou general hospital thessaloniki, greece seemedj 2021, vol 5, no. 2 chronic kidney disease related anemia 56 southeastern european medical journal, 2021; 5(2) 28. study of anemia in chronic kidney disease (ckd) among high-risk hypertensive and diabetic patients in pakistan anemia, diabetes, kidney disease, chronic, hypertension / islamabad, pakistan karachi, pakistan lahore, pakistan 29. observational study of mircera in users of self-application and multidose systems renal anemia of chronic kidney disease device: mircera daun, germany 30. paricalcitol effect on anemia in ckd anemia, chronic kidney disease drug: paricalcitol drug: calcitriol federico ii university naples, italy completed studies conclusion given the importance of chd for public health, it is necessary to raise awareness of chd and encourage early diagnosis and treatment. recommendations and guidelines of all professional nephrology societies are aimed at early diagnosis and timely treatment. when it comes to anemia, the goal is not to correct anemia, but to ensure that the serum hemoglobin level reaches approximately 100 g/l, which can provide the patient with a satisfactory quality of life and minimize the risks related to anemia. the treatment of anemia today represents a largely individualized. it is vital to consider all the current parameters of the disease and condition, the presence of comorbidities, the current method of treatment, as well as the patient’s preference. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare references 1. glogowski t, wojtaszek e, malyszko j. biomarkers of iron metabolism in chronic kidney disease, int urol nephrol. 2021; 53(5):935–944, doi: 10.1007/s11255-020-02663-z 2. honda h, hosaka n, ganz t, shibata t. iron metabolism in chronic kidney disease patients, nakanishi t, kuragano t (eds): ckd-associated complications: progress in the last half century. contrib nephrol. 2019; 198:103–111. https://doi.org/10.1159/000496369 3. gafter-gvili a, schechter a, rozen-zvi b. iron deficiency anemia in chronic kidney disease, acta haematol 2019; 142:44–50, https://doi.org/10.1159/000496492 4. pradeep a, vecihi b, aronoff gr, lyngby mulloy l, talavera f, verrelli m. chronic kidney disease, what are the stages of chronic kidney disease (ckd)?, https://emedicine.medscape.com/article/238 798-overview, jul 21, 2021 5. anil ka. iron metabolism and management: focus on chronic kidney disease. clin investig 2021; 11(1):p46-58. https://doi.org/10.1016/j.kisu.2020.12.003 6. ganz t. anemia of inflammation. n engl j med 2019; 381:1148-57. doi: 10.1056/nejmra1804281 seemedj 2021, vol 5, no. 2 chronic kidney disease related anemia 57 southeastern european medical journal, 2021; 5(2) 7. rački s, bašić-jukić n, kes p, ljutić d, lovčić v.prkačin i, radić j, vujičić b, bubić i, jakić m, belavić ž, šefer s, pehar m, klarić d, gulin m. liječenje anemije u kroničnoj bubrežnoj bolesti – stav hrvatskog društva za nefrologiju, dijalizu i transplantaciju i osvrt na preporuke kdigo i erbp, acta med croatica. 2014; 68:215-221. 8. hill nr, fatoba st, oke jl, hirst ja, o'callaghan ca, lasserson ds, hobbs fd. global prevalence of chronic kidney diseasea systematic review and meta-analysis, plos one. 2016; 11(7) jul 6. doi: 10.1371/journal.pone.0158765 9. locatelli f, mandolfo s, menegato adorati m, villa g, tarchini r, pizzarelli f, conte f, guastoni c, ricciardi b, crotta a. efficacy and safety of once-monthly continuous erythropoietin receptor activator in patients with chronic renal anemia, j nephrol. 2013; 26(6): 1114-1121, doi: 10.5301/jn.5000251 10. eckardt k-u, casadevall n. pure red‐cell aplasia due to anti‐erythropoietin antibodies. nephrol dial transplant. 2003; 18(5):865–869. https://doi.org/10.1093/ndt/gfg182 11.cordina lr, celermajer rd. chronic cyanosis and vascular function: implications for patients with cyanotic congenital heart disease. cardiol young. 2010; 20(3):242-53. doi:10.1017/s1047951110000466 12. feehally j, floege j, tonelli m, j. johnson r. comprehensive clinical nephrology, e-book, sixth edition, elsevier inc 2019, macdougall ic, eckardt k-u., section xvi, chronic kidney disease and the uremic syndrome, chapter 82, anemia in chronic kidney disease pg. 958-965. 13. macdougall ic. cera (continuous erythropoietin receptor activator): a new erythropoiesis-stimulating agent for the treatment of anemia. curr hematol rep. 2005; 4(6):436-40. 14. goldsmith d, covic a. time to reconsider evidence for anaemia treatment (treat) essential safety arguments (esa), nephrology dialysis transplantation. 2010; 25(6):1734-1737. https://doi.org/10.1093/ndt/gfq099 15. j.lamb e., s. levey a., e. stevens p., the kidney disease improving global outcomes (kdigo) guideline update for chronic kidney disease: evolution not revolution. clinical chemistry. 2013; 59(3):462-465. https://doi.org/10.1373/clinchem.2012.184259 16. portolés j, martínl, broseta jj, cases a.anemia in chronic kidney disease: from pathophysiology and current treatments, to future agents. front med (lausanne) 2021; 8:642296. doi:10.3389/fmed.2021.642296. 17. akizawat, yamaguchiy, otsuka t, reusch m. a phase 3, multicenter, randomized, two-arm, open-label study of intermittent oral dosing of roxadustat for the treatment of anemia in japanese erythropoiesis-stimulating agentnaïve chronic kidney disease patients not on dialysis. nephron. 2020; 144(8):372-382, doi: 10.1159/000508100. 18. https://clinicaltrials.gov/ct2/home . author contribution. acquisition of data: hrvačić m, penava m, juras a administrative, technical or logistic support: hrvačić m, penava m, juras a analysis and interpretation of data: hrvačić m, penava m, juras a conception and design: hrvačić m, penava m, juras a critical revision of the article for important intellectual content: hrvačić m, penava m, juras a drafting of the article: hrvačić m, penava m, juras a final approval of the article: hrvačić m, penava m, juras a obtaining funding: hrvačić m, penava m, juras a provision of study materials or patients: hrvačić m, penava m, juras a seemedj 2019, vol 3, no. 2 assessment of nutritional status of elderly people 1 southeastern european medical journal, 2019; 3(2) original article assessment of nutritional status of elderly people in a family medicine practice in relation to mna test, comorbidity and chronic therapy 1 zvonimir bosnić 1, marko miškić 2, karolina veselski 3, domagoj vučić 3, ljijana trtica majnarić 2 1 health centre slavonski brod, croatia 2 faculty of medicine, josip juraj strossmayer university of osijek, osijek, croatia 3 general hospital dr josip benčević, slavonski brod, croatia corresponding author: zvonimir bosnić, zbosnic191@gmail.com received: aug 30, 2019; revised version accepted: dec 5, 2019; published: dec 16, 2019 keywords: nutritional assessment, comorbidity, polypharmacy abstract introduction: nutrition assessment is one of the biggest challenges in family medicine practice because of the increasing number of older people with more comorbidities and chronic therapy. the mna (mini nutritional assessment) test has proven to be the most sensitive and exact tool for this type of research. the aim of this study was to assess whether there is a difference in the nutritional status of elderly people, over 65 years of age, with respect to sociodemographic characteristics, number and type of chronic diseases, and number of medications used in chronic therapy. materials and methods: research subjects were 207 patients at the age of 65 treated at the medical centre slavonski brod during a period of 3 months. during the visit, the nutritional status was examined by using the mna test. the information on chronic diseases and number of medications the patients were using has been collected from the medicus computer program. results: according to the mna test results, 62 subjects (30%) showed risk of malnutrition, average age of the subjects was 72 years and the median of mna test results was 25.50. also, multimorbidity is present in 64.73% of the subjects and 42% of them take more than three medications in chronic therapy. no statistically significant difference was found in the results of the mna test regarding the number of chronic diseases (p =0.89) and number of medications (p = 0.87). conclusion: it is important to regularly monitor the nutritional status in order to prevent progression of chronic diseases and reduce additional cost of treatment. (bosnić z, miškić m, veselski k, vučić d, trtica majnarić lj. assessment of nutritional status of elderly people in a family medicine practice in relation to mna test, comorbidity and chronic therapy. seemedj 2019; 3(2); 1-10) seemedj 2019, vol 3, no. 2 assessment of nutritional status of elderly people 2 southeastern european medical journal, 2019; 3(2) introduction according to the latest epidemiological data, there is a negative demographic trend in the republic of croatia that is evident by the increased number of older people (1,2). demographic aging has affected the republic of croatia, with particular focus on people over 65 years of age who represent the fastest-growing population segment in the world (3,4). according to the 2011 population census data for the republic of croatia, 24.1% persons were older than 60 and 17.7% of persons were over 65 years of age. according to the world health organization (who), the republic of croatia is among the countries with a high percentage of population over 65 (3). according to data of the croatian institute for public health (hzjz), life expectancy for elderly people has increased from 71.0 years in total (66.1 for men, 76.2 years for women) in the year 1991, to 77.9 years in total (74.7 for men, 81 for women) in the year 2014 (4). with the increase of the percentage of older population, there is also a significant increase in the number of medical examinations, consumption of medicinal products and the number of hospitalizations, which requires better socioeconomic awareness and new strategies in treatment of patients. aging weakens the integrative functions of the endocrinological and immune systems. it also causes the accumulation of free radicals, which, in turn, leads to morphological and functional changes in the body and shortens the cellular life span. morphological changes are observable in all organs. these changes are characterized by reduction in cell volume, atrophy and progressive loss of cells (5, 6, 7). also, changes in the cardiovascular system imply lower heart rate, decrease in catecholamine sensitivity and in myofibril contractility (6, 7). there are also changes in the respiratory function, which are characterized by an increased residual volume, decreased parenchyma elasticity and higher risk of developing atelectasis. chronic disease is defined by who as a longlasting and slow-progressing condition. today it is the most common and most expensive health care problem. there are numerous factors which contribute to its development. these are, in addition to the medical ones, socioeconomic factors and political, cultural and environmental aspects (8). therefore, comorbidity represents one of the greatest challenges in 21st century medicine, because it describes the simultaneous occurrence of two or more diseases. this presents a challenge in both scientific research and daily clinical practice and treatment of patients. sometimes treatment for one disease leads to the appearance or exacerbation of another disease, including potentially a fatal outcome and premature death. furthermore, the presence of more diseases and conditions concurrently is directly related to higher mortality, deficient functioning, lower quality of life and more frequent hospitalizations. malnutrition can be defined as a nutrient imbalance in which the intake of energy and other nutritional factors is lower than their consumption. this leads to the development of chronic diseases. malnourished patients are reported to be prone to infections, which lead to prolonged hospitalizations and increased costs of healthcare (10, 11). also, numerous diseases of different organ systems can lead to malnutrition. this means that malnutrition is caused by changes in intake, digestion or absorption of food but also by changes in the metabolism and excretion and/or metabolic requirements for energy, protein and other nutrients (12). in addition, malnutrition can also be a result of low socioeconomic level, inadequate nutritional support as well as the nature of the disease itself (loss of appetite in various acute and chronic diseases). older age is associated with the presence of chronic diseases. this, due to various pathophysiological mechanisms, such as an increased degree of chronic inflammation and oxidative stress, leads to muscular wasting, but also to other changes related to malnutrition (e.g. loss of some micronutrients). multimorbidity, malnutrition and muscular wasting (sarcopenia) increase the risk of negative health outcomes, such as seemedj 2019, vol 3, no. 2 assessment of nutritional status of elderly people 3 southeastern european medical journal, 2019; 3(2) hospitalizations, dependence on others to perform daily activities and premature death (13). important indicators of nutritional status are biochemical methods based on blood and urine tests, where serum protein levels are an especially important factor. albumin is the most prominent in the serum proteins and also the most cited biochemical parameter mentioned in literature as an indicator of malnutrition (14, 15). questionnaires for nutrition status testing are the most useful tools because they are more comprehensive than other measures (based on multiple measured parameters) used in the assessment. furthermore, the literature describes many different questionnaires that have been used so far in clinical practice. the mna-sf (mini nutritional assessment short form) test is a widely used screening tool designed to examine the nutritional status of the elderly, whether they are hospitalized, housed in nursing homes or living independently in a community. this test is used by healthcare professionals and it is based on recording the bmi, food intake, weight loss in the last three months, mobility and the presence of psychological stress and neuropsychological problems in the last three months. screening of the elderly for malnutrition is easier to carry out in general/family medicine practice because it is easier retrieve general population data there. for this purpose, simple but comprehensive and accurate screening tools are required. the mna test has proven to be one of the most appropriate tools (16, 17). the aim of this study was to assess whether there is a difference in the nutritional status of the elderly, at the age of 65 or over, with respect to sociodemographic status, number and type of chronic diseases, number of medications in chronic therapy and the achievement on the mna test. patients and methods the study was conducted on 207 patients (n=207), aged 65 and over, who were treated at the family medicine practice in the health centre of slavonski brod (dom zdravlja slavonski brod). this was done during a period of three months (from january to march 2019). research subjects were treated for various reasons, except those listed as exclusion criteria (patients suffering from acute illnesses and sudden health problems, patients with malignancies, patients with limb amputations, patients examined during home visits, patients on chronic haemodialysis program). when it comes to gender, 90 of the research subjects were men (n=90, 43.47%) and 117 were women (n= 117, 56.52%). a medicus computer program was used to obtain information on chronic diseases and the number of medications in chronic therapy. during the patients’ ambulatory visit, a nutritional assessment was performed using the mna test. the longer version of this test was used. it consisted of 18 questions, which examined patients’ eating habits, type of food they consume, the number of medications in chronic therapy and patients’ personal opinion about their own health condition. the maximum number of points that could be scored in the mna test was 30. the score ranging from 24 to 30 points indicates a good degree of nutrition, 17 to 23.5 indicates a risk of malnutrition and less than 17 points indicates malnutrition (16, 17). clinical characteristics of patients are presented descriptively (categorical variables as absolute and relative frequency, and numerical variables as mean and standard deviation and as interquartile range and median). the chi-square test was used to examine the difference between categorical variables. fischer's exact test was also used. all p-values are two-sided. the significance level will be set to alpha = 0.05. the ibm spss 23 software package (ibm corp. (2015) ibm spss statistics for windows, version 23.0. armonk, new york: ibm corporation) was used for statistical analysis. seemedj 2019, vol 3, no. 2 assessment of nutritional status of elderly people 4 southeastern european medical journal, 2019; 3(2) results according to the mna test results, 62 patients (30%) showed malnutrition risk and only 2 patients (0.9%) had malnutrition. personal history of 134 subjects (64.73%) showed they suffered from more than three chronic diseases and 87 subjects (42%) took more than three medications (table 1). table 1. descriptive data of nutritional status (number and relative frequency) of the categorical variables used in the study n relative frequency gender male 90 43.47% female 117 56. 52% nutritional status well-nourished 143 69.1% risk of malnutrition 62 30% malnutrition 2 0.9% stage 4 2 1% number of chronic diseases < 3 73 35.26% ≥ 3 134 64.73% number of drugs in chronic therapy < 3 120 58% ≥ 3 87 42% the average age of the research subjects was 72 years (interquartile range 69 to 76 years) and the median mna test result was 25.50 (interquartile range 23 to 27). overall, both genders had three or more chronic illnesses present. hypertension was most prevalent as a chronic disease in both genders. musculoskeletal disorders were more common in women, especially osteoporosis. diabetes and copd were equally present in both genders (table 2). table 2. descriptive data (absolute and relative frequencies) on the type and number of chronic diseases depending on gender number of respondents male female n relative frequency n relative frequency > 3 chronic diseases 57 63.33% 77 65.8% < 3 chronic diseases 33 36.7% 40 34.2% diabetes 17 18.9% 22 18.8% hypertension 79 87.8% 108 92.3% gastrointestinal diseases 27 30% 45 38.5% copd 7 7.8% 6 5.1% musculoskeletal disorders 34 37.8% 61 52.1% osteoporosis 1 1.1% 20 17.1% arthritis 11 12.2% 19 16.2% seemedj 2019, vol 3, no. 2 assessment of nutritional status of elderly people 5 southeastern european medical journal, 2019; 3(2) the analysis of nutrition with respect to gender, showed a lack of significant difference in the frequency of nutrition status, the result would be somewhat different if the achievement in the mna test was measured by the average number of points, rather than the categories of malnutrition. accordingly, the results in the student's t-test show a significant difference between the genders (t (205) = 2.40, p = 0.02), with men having a higher score (m = 25.41, sd = 2.77) than women (m = 24.38, sd = 3.24). these results are shown in figure 1. figure 1. arithmetic mean of mna scores and associated confidence intervals (95%) in relation to gender male female there was no difference in nutrition when taking into consideration the number of chronic most of the men who are suffering from three or more diseases, showed good nutritional status. on the other hand, further difference in nutritional status was examined regarding the presence of a single chronic disease (hypertension, musculoskeletal diseases, copd, osteoporosis, arthritis). no significant statistical difference was observed in the distribution of frequencies of a specific chronic disease between well-nourished patients and patients at risk of malnutrition. when it comes to examining the nutritional status of older women, regarding the number of chronic diseases, it should also be mentioned that most women suffering from three or more diseases showed good nutritional status. the nutritional status with respect to the presence of an individual disease did not show a significant statistical difference in the nutritional status between well-nourished elderly women and women at risk of malnutrition. apart from gender, differences in nutritional status were examined considering the age factor. subjects were divided into four categories according to their age; 65 to 69, 70 to 72, 73 to 76 and 77 to 91 years of age. the first age group (65-69 years) was the one with the highest percentage of three or more chronic diseases (table 3). seemedj 2019, vol 3, no. 2 assessment of nutritional status of elderly people 6 southeastern european medical journal, 2019; 3(2) table 3. distribution of respondents by age categories and nutritional status p -statistical significance, * chi-square test, † fisher's exact test examining the difference in nutritional status with regard to the number of medications a subject was taking, the chi-square had no significan potential direction of the difference, student's ttest was again performed on the mna resu nutritional assessment, comorbidity, polypharmacy lts, showing no significant difference (t (205) = 0.59, p = 0.55). however, upon examining the degree of malnutrition considering patients’ age and score on the mna test, it was observed that well-nourished patients were younger (m = 72.02, sd = 5.38) than malnourished patients (m = 75.64, sd = 6.12) discussion one of biggest challenges for a family medicine doctor is to recognize and prevent malnutrition due to negative demographic situations and a large number of elderly people. also, there are other factors that make this work and the objectivity of clinical judgment more difficult. these include a large number of outpatient examinations, lack of medical staff and working as a substitute in different outpatient clinics. the results obtained in our research showed that elderly people are often diagnosed with two or more chronic diseases. however, the results also show that there is no significant correlation between the number of chronic diseases and nutritional status. it should be emphasized that elderly men and elderly women show signs of multimorbidity equally (table 1). one of the biggest challenges was to choose a proper screening tool for malnutrition (18, 19). reasons for that are multimorbidity and a relatively high number of medications used in number (%) of respondents wellnourished insufficient nutrition total p number of chronic diseases (65 – 69) < 3 22 (33.3) 6 (9.1) 28 0.56* ≥ 3 32 (48.5) 6 (9.1) 38 total 54 12 66 number of chronic diseases (70 – 72) < 3 14 (31.8) 3 (6.8) 17 >0.99† ≥ 3 22 (50) 5 (11.4) 27 total 36 8 44 number of chronic diseases (73–76) < 3 8 (16.7) 5 (10.4) 13 0.73† ≥ 3 24 (50) 11 (22.9) 35 total 32 16 48 number of chronic diseases (77 – 91) < 3 6 (12.2) 9 (18.4) 15 0.79* ≥ 3 15 (30.6) 19 (38.8) 34 total 21 28 49 seemedj 2019, vol 3, no. 2 assessment of nutritional status of elderly people 7 southeastern european medical journal, 2019; 3(2) chronic therapy, which are associated with higher mortality and a lower quality of life. previous research has shown that taking six different medications increases the risk of adverse effects up to 80%. also, taking eight medications increases the risk of medication interactions up to 100% (19, 20). in our research, 87 participants (42%) took more than three medications (table 1). based on the given results, in order to prevent adverse effects, it is highly important to be aware of the number and type of medications used in chronic therapy. this study did not show any statistical difference between the number of medications and the nutritional status of elderly people. in our research, only 62 (30%) participants out of 207 were at risk of malnutrition. these results confirm a good nutritional status of elderly people (table 1). according to the results they achieved in the mna test, research subjects were further examined based on differences in gender and age with regard to their nutritional status. it has been observed that men achieve a slightly higher score on the mna test than women, which means that women are more at risk of malnutrition. these results are shown in figure 1. on the other hand, it should be noted that the data obtained by the mna result indicate a good degree of nutritional status in both cases (16, 17). results of this study do not differ significantly from the results found in the research by kalan u. et al., who studied the connection between the mna test, nutritional status and the potential factors that lead to malnutrition. comparing our results to the results of the study conducted by tavassoli et al., 30% of the results of participants at risk of malnutrition in our research are slightly lower than those for malnutrition according to the mna test (21). in relation to age, we separated our participants into four groups. patients at the age of 77 to 91, who make up 19,. 4% of our research subjects, showed risk for malnutrition. the adverse impact of multimorbidity on nutritional status is more manifested in older age groups (> 77 years), and the number of chronic disease (presence of 3 and > chronic diseases) is also increased (table 3). overall, hypertension was the most prevalent chronic disease in both genders (table 2). when it comes to good nutritional status of elderly people, it is expected that the majority of them suffer from a cardiovascular disease. this can be explained by the generally high prevalence of hypertension in elderly people, where three out of four people suffer from hypertension (19). despite the presence of hypertension in both genders, there was no significant difference in nutritional status between research subjects, when hypertension and number of chronic diseases are taken into consideration. similar results were obtained by ivana platuzic in her research, as a part of her graduate work at the faculty of food technology in osijek (22). when compared to other chronic diseases, musculoskeletal disorders are more common in women (table 2). osteoporosis and arthritis were the disorders that were most presented in our research. these kinds of diseases are associated with degenerative changes and carry the risk of falls. furthermore, it is important to be aware of the development of the physical disability syndrome, also known as the frailty syndrome. it is a common clinical syndrome in older adults that carries an increased risk for poor health outcomes that include disability, hospitalization and mortality. that is why this syndrome is of great interest to scientific circles. despite the greater distribution of osteoporosis in older women, there was no significant connection between nutritional status, presence of musculoskeletal diseases and the number of chronic diseases. however, the presence of musculoskeletal disorders and multimorbidity (> 3 chronic diseases) may have an effect on malnutrition only in the third and fourth age group (>73 and >77 years, respectively). furthermore, in future research, more attention should be paid to the diagnosis of painful back. this is important because more and more people perform sedentary work, which, over time, leads to the development of painful back. moreover, it is important to make a stratification of diagnosis of painful back by the severity of the seemedj 2019, vol 3, no. 2 assessment of nutritional status of elderly people 8 southeastern european medical journal, 2019; 3(2) symptoms and the clinical manifestation in order to evaluate degenerative changes. in our research, occurrence of diabetes mellitus is very similar in both genders (table 2). moreover, there was no significant difference in nutritional status between men and women if we take into consideration the presence of diabetes and the number of chronic diseases. ursulin-trstenjak et al. studied the relationship between obesity, hypertension and diabetes mellitus in elderly people. they have tried to prove a direct link between obesity and chronic diseases (23). in previous studies, malnutrition was evident in situations where chronic respiratory diseases were present (24). according to the small number of participants who suffered from chronic respiratory diseases, the data obtained showed a similar distribution of disease in both genders, and there was no significant statistical difference between nutritional statuses. in future research, for a better understanding of the connection between chronic respiratory diseases and nutritional status, it is important to have more research subjects involved. even though the previous studies have proven there is a connection between malnutrition and chronic gastrointestinal diseases, the results of this study showed no significant difference in the nutritional status of elderly men and women. while we did not isolate gastrointestinal diseases as specific factors, in future research for better understanding of nutritional status, it will be useful to isolate participants who suffer from inflammatory bowel disease and multimorbidity. the aim of this study was also to find a connection between age and nutritional status. obtained data found a significant difference in the degree of nutrition with respect to age. it was observed that patients with normal nutrition were younger than those with poor nutritional status. even though there are many available screening tools for conducting research, the mna test is still the most efficient one, because of its simplicity. according to the results of this crosssectional study, as well as the results of previous studies, we can state that mna is the optimal, accessible ambulatory tool for conducting research in family medicine practice. this research has some limitations as it was conducted only in the health centre in slavonski brod. for more accurate data, it would be best to extend this research to other health centres in other counties and compare the results. one of the biggest problems that will affect family medicine practice in the next 40 years is an increased number of people over 60 years of age. it is expected that the number of elderly people will increase by 50%. therefore, in future research, more attention should be focused on a comprehensive approach to nutrition monitoring in elderly people. to conclude, it is important to educate elderly people and raise their awareness of their own nutritional status in order to prevent malnutrition and improve their quality of life. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. . references 1. the census of population, households and dwellings 2011. croatian bureau of statistics, zagreb, 2011. available at: http://www.dzs.hr/. accessed: 13 june 2019. 2. croatian health statistics yearbook 2017. available at: https://www.hzjz.hr/wpcontent/uploads/2019/03/ljetopis_201 7.pdf. accessed: 13 june 2019. 3. nejašmić i, toskić a. starenje stanovništva u hrvatskojsadašnje stanje seemedj 2019, vol 3, no. 2 assessment of nutritional status of elderly people 9 southeastern european medical journal, 2019; 3(2) i perspektive. croatian geographical bulletin, 2013;75/1, 89 –110. 4. croatian institute of public health. usporedba pokazatelja o vodećim javnozdravstvenim problemima u republici hrvatskoj i europskoj uniji. available at: https://www.hzjz.hr/wpcontent/uploads/2017/01/pokazatelji_r h_eu.pdf. accessed: 13 june 2019. 5. damjanov i, jukić s, nola m. patologija, 3rd reworked and supplemented edition. zagreb: medicinska naklada; 2011. pp. 4142. 6. vrndić o, kostic i. patofiziološki mehanizam procesa starenja. medicinski časopis. 2010;3, 30-36. 7. gamulin s, marušić, m. patofiziologija, 7th updated and modified edition. zagreb: medicinska naklada. 2011; pp. 2427. 8. vukelić m. percepcija bolesti i terapije u bolesnika s kroničnim bolestima. [graduate thesis]. zagreb: faculty of pharmacy and biochemistry. 2016. 9. perko g, tomekroksandić s, mihok d, puljak a, radašević h, tomić b. vodeći multifaktorijalni gerijatrijski sindrom u starijih osoba ("4n" u gerijatriji). hrvatski časopis za javno zdravstvo. 2006; 20-22. 10. krznarić ž. klinička prehrana u gastroenterologiji. medicus [internet]. 2006 [accessed: 18 august 2019];15(1_gastroenterologija):169-181. available at: https://hrcak.srce.hr/18845. 11. vranešić bender d, krznarić ž. malnutrition undernutrition of hospital patients. medicus. 2008. volume 17, 7179. 12. jeejeebhoy kn. hospital malnutrition: is a disease or lack of food? clin nutr. 2003;22: 219. 13. haehling s von. the wasting continuum in heart failure: from sarcopenia to cachexia. proceedings of the nutrition society. 2015;74:367–377. 14. fuhrman mp, charney p, mueller cm. hepatic proteins and nutrition assessment. j am diet assoc. 2004;104:1258-64. 15. fuhrman mp. the albumin-nutrition connection: separating myth from fact. nutrition. 2002;18:199-200. 16. kalan u, arik f, isik at, soysal p. nutritional profiles of older adults according the mini-nutritional assessment. aging clin exp res. 2019. 17. soysal p, veronese n, arik f, kalan u, smith l, isik at. mini nutritional assessment scale-short form can be useful for frailty screening in older adults. clin interv aging. 2019;14:693-699. 18. hien h, berthé a, drabo mk, meda n, konaté b, tou f i sur. prevalence and patterns of multimorbidity among the elderly in burkina faso: cross-sectional study. trop med int health. 2014;19 (11):1328-33. 19. katić m, švab i et al. obiteljska medicina. zagreb 1st edition. alfa; 2013. 20. zadak z, hyspler r, ticha a, vlcek j. polypharmacy and malnutrition. current opinion in clinical nutrition & metabolic care. 2013;16 (1):50-55. 21. tavassoli n, guyonnet s, abellan van kan g, sourdet s, krams t, soto mf et al. description of 1,108 older patients referred by their physician to the “geriatric frailty clinic” for assessment of frailty and prevention of disability at the gerontopole. j nutr health aging. 2014;18:457-64. seemedj 2019, vol 3, no. 2 assessment of nutritional status of elderly people 10 southeastern european medical journal, 2019; 3(2) 22. platužić ivana. procjena statusa uhranjenosti starijih osoba. [graduate thesis]. osijek: faculty of food technology osijek. josip juraj strossmayer university of osijek, 2018 23. uršulin-trstenjak n, levanić d, hasaković-felja m. pretilost kao faktor rizika za nastajanje kardiovaskularnihkoronarnih bolesti. tehnički glasnik. 2015,9:230-234. 24. ezzell l, jensen gl. malnutrition in chronic obstructive pulmonary disease. the american journal of clinical nutrition, volume 72, issue 6. 2000;1415– 1416. seemedj 2020, vol 4, no. 2 histopathological findings of operated tumors of the parathyroid glands 113 southeastern european medical journal, 2020; 4(2) original article the histopathological findings of operated tumors of the parathyroid glands and patient data: a single centre experience 1 ivan feldi *1,2, ana jurić 1, ksenija marjanović 1,3, hrvoje mihalj 1,4, tatjana bačun 1,5 1 faculty of medicine, osijek, croatia 2 department of internal medicine, general county hospital našice, našice, croatia 3 clinical department of pathology and forensic medicine, university hospital centre osijek, osijek, croatia 4 department of otorhinolaryngology, head and neck surgery, university hospital centre osijek, osijek, croatia 5 department of endocrinology, university hospital centre osijek, osijek, croatia *corresponding author: ivan feldi, ivanfeldi2@gmail.com received: mar 4, 2020; revised version accepted: may 22, 2020; published: nov 12, 2020 keywords: parathyroid glands, primary hyperparathyroidism, histopathology, staining and labeling abstract introduction: parathyroid proliferative disorders include adenoma, hyperplasia and carcinoma. adenoma and hyperplasia are more commonly found in women, while carcinoma, which is very rare, is equally common in both sexes. the aim of this study was to analyze parathyroid tumors location and histopathology and to compare differences between the sexes. tumors were surgically removed at the university hospital centre osijek between 2016 and 2019. patients and methods: patients of both sexes who underwent parathyroidectomy for parathyroid tumor at the university hospital centre osijek between 2016 and 2019 were included in the study. parathyroid tumor samples were histologically analyzed, and their size and histopathology were noted. existing documentation on patients with parathyroid tumor was used. analyses were done on archived histologic material stained with hematoxylin and eosin. results: overall, 19 samples of parathyroid tumor were included in this study. tumors of the parathyroid glands were most commonly localized on the lower left parathyroid (8 cases, 42%), and least commonly on the upper right parathyroid (2 cases, 11%). the most common disorder was adenoma (11 cases, 58%), followed by hyperplasia, while no cases of cancer were diagnosed. women underwent tumor operations more frequently than men (17 versus 2 cases). there was no correlation between histopathology of parathyroid tumor and sex (fisher’s exact test, p = 1) or between tumor location and sex (fisher’s exact test, p = 1). conclusion: the most common locations of tumors of the parathyroid glands were the inferior glands; the most common location was the left inferior parathyroid gland and the most common disorder was adenoma. women were operated on more frequently than men. (feldi i, jurić a, marjanović k, mihalj h, bačun t. the histopathological findings of operated tumors of the parathyroid glands and patient data: a single centre experience. seemedj 2020; 4(2); 113-120) seemedj 2020, vol 4, no. 2 histopathological findings of operated tumors of the parathyroid glands 114 southeastern european medical journal, 2020; 4(2) introduction parathyroid glands are 4 nodular structures located behind every pole of the thyroid gland. they are separated from thyroid gland tissue by a fibrous capsule (1). their size is mostly around 5 x 3 x 1 mm, and they weigh up to 50 mg (2). on rare occasions, the number of parathyroid glands can vary from 1 to 12 parathyroid glands (3). due to embryonic development, inferior parathyroid glands are most frequently located ectopically. common ectopic locations are within the thymus, the mediastinum and the thyroid gland, but they are also rarely found within the vagus nerve, the pharynx and the esophagus (4). the main function of parathyroid glands is calcium homeostasis. parathyroid hormone increases serum calcium by increasing renal tubular reabsorption of calcium, bone resorption and by indirectly increasing vitamin d levels, which leads to increased intestinal calcium absorption. parathyroid hormone excretion is regulated by ionized calcium levels, extracellular phosphate levels and vitamin d (5). chief cells, adipocytes and oxyphil cells make up the parathyroid gland parenchyma. chief cells produce and secrete parathyroid hormone (6). oxyphil cell function is not fully understood. they are considered to secrete parathyroid hormone in secondary parathyroid hyperplasia (7). hematoxylin and eosin stain is used to observe parathyroid gland structure, while immunochemistry is used to identify molecular markers (8). parathyroid gland dysfunction can have different causes. increased secretion of parathyroid hormone from at least one parathyroid gland leads to primary hyperparathyroidism. it is mostly caused by a single adenoma, but can be caused by multiple adenoma, hypertrophy of all 4 parathyroid glands, and rarely by carcinoma (9). secondary hyperparathyroidism is a condition of parathyroid hormone oversecretion in response to abnormally low calcium in the blood due to other pathological processes, such as kidney failure, gastrointestinal malabsorption, or vitamin d deficiency. parathyroid hyperplasia caused by prolonged hypocalcemia, which is seen mostly in chronic kidney failure, is defined as tertiary hyperparathyroidism (10, 11). hypoparathyroidism is a rare disorder that occurs after damage to or removal of parathyroid glands, for example during thyroidectomy (12). pseudohypoparathyroidism occurs when the body, and especially the kidneys, develops resistance to the parathyroid hormone (13). parathyroid proliferative disorders include adenoma, hyperplasia and carcinoma. these usually manifest as primary hyperparathyroidism (2). parathyroid adenoma usually occurs between the ages of 50 and 70. microadenomas usually do not have a capsule. larger adenomas have a thin fibrous capsule and can have cystic degeneration in the tumor. their average weight is one gram. macroscopically, the adenoma is smooth, soft and reddish-brown in color, in contrast to the yellow-brown of a normal parathyroid gland (2). there may be normal parenchyma of a parathyroid gland around the edge. histologically, there is a population of enlarged cells, glandular architecture with secretions, atrophy or compression of parathyroid gland tissue (14, 15). parathyroid hyperplasia usually occurs as a result of calcium deficiency, mostly due to chronic kidney disease. all 4 parathyroid glands are enlarged. in certain cases where glands are unevenly enlarged, they may be mistaken for adenoma. resection will not resolve the problem in such case (2). histologically, there is parenchymal fat accumulation and nodular distribution of chief and oxyphil cells (14, 15). parathyroid carcinoma is mostly seen in middleaged patients. risk factors include hereditary syndromes such as men 1 and men 2 (multiple endocrine neoplasia type 1 and type 2), familial isolated hyperparathyroidism, neck radiation and chronic kidney disease. parathyroid hormone and calcium are more increased than in adenoma. in addition to the usual hypercalcemia symptoms such as nausea, vomiting, ulcers, pancreatitis, depression and seemedj 2020, vol 4, no. 2 histopathological findings of operated tumors of the parathyroid glands 115 southeastern european medical journal, 2020; 4(2) languor, there are also special symptoms of the disease, such as osteoporosis, subperiosteal bone resorption and pathological fractures. using ultrasound, carcinoma is identified as a mass, usually in inferior poles of the thyroid, with a size greater than 3 cm (16, 17). histologically, carcinomas look very similar to adenomas, which is why it is difficult to diagnose the disease with only histological analysis. histological features of parathyroid carcinoma are thick fibrous capsule, adherence to surrounding tissue, vascular and capsular invasion, necrosis and increased mitotic activity (18). immunohistochemical methods help differentiate carcinoma from adenoma. the only unequivocal criterion for carcinoma are metastases (16). ultrasound and technetium tc 99m sestamibi scintigraphy scan are most commonly used for localization of parathyroid tumor. spect (single-photon emission computed tomography) allows a 3-dimensional image. scintigraphy and spect combined have up to 98% sensitivity for detection of parathyroid adenoma (2). detection of parathyroid glands by imaging methods is of no importance for primary hyperparathyroidism confirmation. other localization techniques include mri (magnetic resonance imaging), ct (computerized tomography) and venous parathyroid hormone sampling (19). parathyroidectomy is the only definitive treatment of primary hyperparathyroidism. observation and pharmacological therapy are less effective and less cost-effective than surgery, even in asymptomatic patients (19 21). according to the guidelines of the american association of endocrine surgeons, parathyroidectomy is indicated in all patients with symptomatic primary hyperparathyroidism; when the serum calcium level is more than 1 mg/dl (0,25 mmol/l) above normal, regardless of whether objective symptoms are present or absent; when there is objective evidence of renal involvement, including silent nephrolithiasis, nephrocalcinosis, hypercalciuria, or impaired renal function; in patients with primary hyperparathyroidism and osteoporosis; when primary hyperparathyroidism is diagnosed in patients aged 50 or younger; in patients with parathyroid cancer; in patients who are unable or unwilling to comply with observation protocols; in patients with neurocognitive and/or neuropsychiatric symptoms that are attributable to primary hyperparathyroidism. many different surgical methods to streamline parathyroidectomy and reduce the risk of complications have been developed; they are collectively termed minimally invasive parathyroidectomy (mip). the most commonly used auxiliary method that significantly increases surgery success is intraoperative parathyroid hormone measurement (ipm). if, during minimally invasive parathyroidectomy, intraoperative parathyroid hormone measurement indicates residual hypersecreting tissue, conversion to bilateral exploration (be) is necessary. minimally invasive parathyroidectomy has the most success with solitary adenoma (19). preventive measures and routine monitoring of serum calcium, creatinine and bone density are recommended in asymptomatic patients that do not meet the criteria for surgery (22). the aim of this cross-sectional study with historical records was to investigate parathyroid tumors that were surgically removed at the university hospital centre osijek between january 2016 and march 2019. the aim was also to review tumor location and histopathology and to compare differences between sexes. patients and methods patients of both sexes who underwent parathyroidectomy for parathyroid tumor at the university hospital centre osijek between january 2016 and march 2019 were included in the study. data were obtained for 19 patients. in the clinical department of pathology and forensic medicine, parathyroid tumor samples were histologically analyzed; their size and level of histopathological tumor differentiation were noted. existing documentation on patients with parathyroid tumor of the clinical department of pathology and forensic medicine and the department of maxillofacial surgery was used. seemedj 2020, vol 4, no. 2 histopathological findings of operated tumors of the parathyroid glands 116 southeastern european medical journal, 2020; 4(2) analysis was done on archived histologic material stained with hematoxylin and eosin. approval of the institutional ethical committee for this study was obtained from faculty of medicine osijek. statistical analysis statistical analysis was done using medcalc statistical software version 18.9. normal distribution of variables was tested using the kolmogorov-smirnov test. comparisons between nominal variables due to the small number of samples were made using fisher’s exact test. alfa = 0.05 was set as the level of statistical significance. results data were obtained for 19 patients diagnosed with parathyroid tumor. in regard to histopathology, the studied sample included only adenoma and hyperplasia. women underwent parathyroid tumor operations more often than men. of total of 19 samples, there were 2 (11%) male samples, and 17 (89%) female samples. table 1. parathyroid tumor location in both sexes right inferior parathyroid gland left inferior parathyroid gland right superior parathyroid gland left superior parathyroid gland female 5 (26%) 8 (42%) 2 (11%) 2 (11%) p = 1 * male 1 (5%) 0 0 1 (5%) total 6 (31%) 8 (42%) 2 (11%) 3 (16%) * fisher’s exact test parathyroid tumor location was observed. table 1 shows that parathyroid tumors were predominantly located on inferior parathyroid glands (14 cases, 74%) in comparison with superior parathyroid glands (5 cases, 26%). the most common location of the tumor was the left inferior parathyroid (8 cases, 42%). there were 6 cases (31%) of right inferior, 3 cases (16%) of left superior and 2 cases (11%) of right superior parathyroid gland involvement. of the 2 obtained samples of parathyroid tumor in men, one was located on the right inferior and one on the left superior parathyroid gland. in regard to histopathology, one sample was of hyperplasia, and the other was of parathyroid adenoma. table 2. distribution of parathyroid gland tumor in both sexes by histopathological diagnosis adenoma hyperplasia undetermined disorders of parathyroid tissue female 10 (53%) 3 (16%) 4 (21%) p = 1 * male 1 (5%) 1 (5%) 0 total 11 (58%) 4 (21%) 4 (21%) * fisher’s exact test in our research, we examined the distribution of parathyroid tumor by histopathological diagnosis. the most common parathyroid tumor was adenoma. in regard to histopathology, 11 samples (58%) were determined as adenoma, 4 samples (21%) as hyperplasia and 4 samples (21%) as undetermined disorders of parathyroid tissue (table 2). in four cases termed as “undetermined disorders of parathyroid tissue”, due to damaged samples, it was not clear whether the tissue histopathology matched adenoma or hyperplasia. seemedj 2020, vol 4, no. 2 histopathological findings of operated tumors of the parathyroid glands 117 southeastern european medical journal, 2020; 4(2) although women exhibited more adenoma compared to hyperplasia than men, there is no correlation between histopathology of parathyroid tumor and sex (fisher’s exact test, p = 1). likewise, even though there were more inferior parathyroid tumors in regard to superior in women when compared to men, there is also no correlation between tumor location and sex (fisher’s exact test, p = 1). statistical analysis is not valid because the male data sample was small. discussion parathyroid tumors include adenoma, hyperplasia and carcinoma. they most often present as primary hyperparathyroidism. parathyroidectomy is the only definitive treatment. from 1925, when felix mandl performed the first successful parathyroidectomy in vienna, many surgical techniques have been developed that have become less and less invasive (23). minimally invasive parathyroidectomy is a relatively new technique that is used increasingly commonly in parathyroid adenoma treatment. its success depends on imaging of the tumor. ultrasound, technetium tc 99m sestamibi scintigraphy scan, and intraoperative parathyroid hormone measurement can aid the success of the operation. the most common parathyroid tumor locations are inferior parathyroid glands, with frequency of occurrence from 70 to 86.7% (24 26). in our study, tumor was likewise most commonly located on inferior parathyroid glands. according to our study, the most common parathyroid tumor location was the left inferior parathyroid gland, which was also the case in el-hady’s and usta’s studies (24, 25). minimally invasive parathyroidectomy decreases the risk of postoperative hypocalcemia, the length of procedure, the length of hospital stay and overall treatment cost (27). primary hyperparathyroidism is more often found in women (10). in the conducted study, women were also more affected by parathyroid tumors. the most common parathyroid gland tumor is adenoma (80-85%), which is followed by hyperplasia (15%), and carcinoma, which is extremely rare (2). in our study, a similar distribution was seen, but there were no cases of carcinoma. depending on the literature, carcinoma represents from under 1% to 5% of cases of parathyroid tumor (28-30). diagnosing carcinoma is not simple. there are no special clinical manifestations and histology is ambiguous. clinical manifestations can be more extreme than with other causes of primary hyperparathyroidism (16). atypical parathyroid adenoma or parathyroid neoplasm of uncertain malignant potential is a special histological entity that does not meet all the criteria for carcinoma. its course is more benign, it is less aggressive than parathyroid carcinoma, and it has a greater 5-year survival rate (31, 32). in the same period, three patients had parathyroidectomy for multi-gland hyperplasia due to secondary hyperparathyroidism (results not shown). all three of them had chronic kidney failure and were on a hemodialysis program. in the conducted study, primary hyperparathyroidism was the most common clinical diagnosis, followed by secondary hyperparathyroidism. primary hyperparathyroidism occurs in over 2% of the population older than 55 (33). it is usually caused by solitary adenoma, but can also be caused by multiple adenomas, hypertrophy and carcinoma. risk factors include neck radiation, lithium intake or hereditary factors such as men 1 and men 2 syndromes (8). it manifests as hypercalcemia with increased or normal level of parathyroid hormone. patients usually have no symptoms, but in symptomatic patients, it manifests as fatigue, weakness, depression, gastroesophageal reflux and bone pain. in the advanced stage, it can manifest as skeletal disease, nephrolithiasis and cardiovascular disease (10). in secondary hyperparathyroidism, there is normal parathyroid hormone secretion as a response to chronically low calcium levels. low calcium levels are caused by malabsorption, vitamin d deficiency, kidney failure and taking certain medication, such as thiazide diuretics. serum calcium level is low or normal, and parathyroid hormone level is normal. in tertiary seemedj 2020, vol 4, no. 2 histopathological findings of operated tumors of the parathyroid glands 118 southeastern european medical journal, 2020; 4(2) hyperparathyroidism, after an initial stimulus, parathyroid glands become autonomous, and parathyroid hormone levels and calcium levels resemble primary hyperparathyroidism. clinical symptoms of secondary hyperparathyroidism depend on the underlying cause (8, 34). our study did not show a correlation between sex and tumor location, or between sex and type of tumor. to our knowledge, no study has so far analyzed the correlation between sex and tumor location. only 2 male samples were analyzed, which was not sufficient for making an adequate statistical analysis. corresponding to the correlation between sex and tumor location, it is known that adenoma is found 3 times more often in women than in men (18, 35). in the study by cvasciuc (36), patients with parathyroid adenoma were predominantly female, and there was a similar female-to-male ratio in regard to adenoma and hyperplasia. carcinoma affects both sexes equally commonly (16). it is important to note that these are data collected from patients who were subjected to parathyroidectomy at our university hospital centre. not all patients with primary or secondary hyperparathyroidism are operated on, so this study can in no way present the prevalence of hyperparathyroidism in the area gravitating to the university hospital centre in osijek. the sizes of tumors in samples from this area were 0.7-2.2 cm (results not shown). the main limitation of the study was its small sample, which is probably the reason why there were no cases of carcinoma. if the sample were larger, carcinoma frequency could have been assessed. the aim of this study was to review the possible connection between sex and parathyroid tumor histopathology or location, but only 2 male samples were obtained, which was not sufficient for making a statistical analysis. to conclude, the study shows that women are operated on for tumors of the parathyroid gland more often, which most commonly manifest as adenomas. parathyroid tumors were more frequently located in inferior parathyroid glands, and the most common location was the left inferior parathyroid gland. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. i references 1. taterra d, wong lm, vikse j, sanna b, pękala p, walocha j, cirocchi r, tomaszewski k, henry bm. the prevalence and anatomy of parathyroid glands: a meta-analysis with implications for parathyroid surgery. langenbecks arch surg. 2019; 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18(1):79-95. 35. edafe o, collins ee, ubhi cs, balasubramanian sp. current predictive models do not accurately differentiate between single and multigland disease in primary hyperparathyroidism: a retrospective cohort study of two endocrine surgery units. ann r coll surg engl. 2018; 100(2):140-145. 36. cvasciuc it, ismail w, lansdown m. primary hyperparathyroidismstrategy for multigland disease in the era of spect-ct. acta endocrinol (buchar). 2017;13(1):1–6. doi:10.4183/aeb.2017.1 i author contribution. acquisition of data: feldi i, jurić a, marjanović k, mihalj h, bačun t administrative, technical or logistic support: feldi i, jurić a, marjanović k, mihalj h, bačun t analysis and interpretation of data: feldi i, jurić a, marjanović k, mihalj h, bačun t conception and design: feldi i, jurić a, marjanović k, mihalj h, bačun t critical revision of the article for important intellectual content: feldi i, jurić a, marjanović k, mihalj h, bačun t drafting of the article: feldi i, jurić a, marjanović k, mihalj h, bačun t final approval of the article: feldi i, jurić a, marjanović k, mihalj h, bačun t guarantor of the study: feldi i, jurić a, marjanović k, mihalj h, bačun t obtaining funding: feldi i, jurić a, marjanović k, mihalj h, bačun t provision of study materials or patients; feldi i, jurić a, marjanović k, mihalj h, bačun t statistical expertise: feldi i, jurić a, marjanović k, mihalj h, bačun t other: feldi i, jurić a, marjanović k, mihalj h, bačun t seemedj 2021, vol 5, no. 1 telemedicine in otorhinolaryngology 122 southeastern european medical journal, 2021; 5(1) review article implementation of telemedicine in otorhinolaryngology 1 hrvoje mihalj 1, 2, željko zubčić 1, 2, andrijana včeva 1, 2, željko vranješ 1, 2, josip maleš 1,2, darija birtić 1, 2, tihana mendeš 1, 2, stjepan grga milanković 2, tin prpić 1, 2, vjeran bogović 1, 2, ivan abičić 1, 2, matej rezo 2, miroslav moguš 3, anamarija šestak *2 1 department of otorhinolaryngology and maxillofacial surgery, faculty of medicine, university of osijek, j. huttlera 4, 31000 osijek, croatia 2 department of otorhinolaryngology and head and neck surgery, clinical hospital centre osijek, j. huttlera 4, 31000 osijek, croatia 3 online application development team, a1 hrvatska d.o.o., vrtni put 1, 10 000 zagreb, croatia *corresponding author: anamarija šestak, annamarijasestak@gmail.com received: jan 28, 2021; revised version accepted: mar 12, 2021; published: apr 28, 2021 keywords: telemedicine; otolaryngology; distance counseling abstract telemedicine is a term that covers all procedures supported by communication technology, which has the purpose of providing health services at a specific spatial distance. this is an area that is developing rapidly and has found particular application in otorhinolaryngology, given that a large number of surgeries are performed with the help of endoscopes and microscopes. telemedicine also represents a significant advantage during the coronavirus pandemic, both in terms of treating patients and monitoring them more effectively. for the purpose of preparing this article, research published on scopus, pubmed, google scholar, and google was reviewed using the keywords “telemedicine” and “otorhinolaryngology”. this review article provides a summary and the latest insights in this broad and fast-growing area. the development of telemedicine in croatia as well as a special review of the application of telemedicine during the coronavirus pandemic is also presented in this article. (mihalj h, zubčić ž, včeva a, vranješ ž, maleš j, birtić d, mendeš t, milanković sg, prpić t, bogović v, abičić i, rezo m, moguš m, šestak a. implementation of telemedicine in otorhinolaryngology. seemedj 2021; 5(1); 122-134) seemedj 2021, vol 5, no. 1 telemedicine in otorhinolaryngology 123 southeastern european medical journal, 2021; 5(1) introduction telemedicine is a term that covers all procedures supported by communication technology, which has the purpose of providing health services at a specific spatial distance. the concept of telemedicine includes various activities, such as teleassistance, telemonitoring, teleconsultation, tele-expertise, videoconferencing, and telesurgery (1). these are the most common terms, although standardised terminology is missing due to a very rapid development of telemedicine (2,3). the goal of all these activities is to increase the quality of health care and make health care available to every patient at any place. telemedicine is a rapidly evolving field that has provided much more efficient access to patients. telemedicine has been applied in many specialties, including otorhinolaryngology. for the purpose of preparing this review article on the application of telemedicine in otorhinolaryngology and the latest knowledge in this field, research published on scopus, pubmed, google scholar, and google was reviewed using the keywords “telemedicine” and “otorhinolaryngology”. abstracts of relevant articles were studied and based on relevant data, full texts of the papers were reviewed. considering that telemedicine in otorhinolaryngology is heterogeneous, the review article is divided into several parts depending on the field of otorhinolaryngology concerned. history of telemedicine and its development in croatia in the 1960s, nasa sent humans into space as part of the apollo project. astronauts should be medically protected in different conditions – this was the beginning of biomedical observation development (4-7). as there was a need for connection and astronauts’ mutual education, nasa played a significant role in telemedicine development due to its ability of establishing satellite links. the first complete telemedicine system was installed in 1967, connecting the boston airport medical station with the massachusetts general hospital. in 1989, nasa conducted the first international telemedicine project, space bridge to armenia, following a strong earthquake in armenia in 1988 (8). the first steps in the development of telemedicine in croatia were made in the 1980s, when an ecg was recorded based on a distant patient’s first message at the clinic for cardiovascular diseases in zagreb. the cardiologist received the imaging record and interpreted it. the technology of the time was not able to transmit mutual, simultaneous change of image and sound. progress was made after the development of isdn networks and establishment of the government agency croatian academic and research network (carnet) in 1991/1992. in 1993, a national telepathology system with remote hospital centres was established in zagreb. in 1995, a program was launched to monitor people with diabetes across the country. in the following year, a telecommunication program for cardiac electrostimulation was presented, and in 1998, the national teleradiology and teleneurosurgery programs were in full function. in the same year, the croatian academy of medical sciences organised a scientific conference on croatian telemedicine achievements to encourage cooperation between outpatient facilities and higher health system levels. three years later, central institutions made connections with some islands. during this period, large medical centres developed numerous tele-education programs (9). almost every clinical specialty uses telemedicine in a certain way, but some specialties use it to a greater extent than others. radiologists, for example, use telemedicine extensively. the teleradiology system electronically transmits radiological images and image descriptions. by transferring the image to the radiologist, he can interpret the radiological images very quickly. after nasa, another essential factor in the development of telemedicine was the military. in the academic year 2009/2010, the faculty of medicine in osijek recognised the importance of knowing the basics of telemedicine, thus seemedj 2021, vol 5, no. 1 telemedicine in otorhinolaryngology 124 southeastern european medical journal, 2021; 5(1) introducing it as a course in the compulsory elective module (clinical medicine) as part of the postgraduate doctoral study programme of biomedicine and health. the course consists of 18 lecture classes and 12 seminar classes and over 40 students enrolled in this elective course in the last two generations. the timeline of the development of telemedicine in croatia and the world is shown in figure 1. figure 1. timeline of telemedicine development in croatia and the world potential of telemedicine development with the development of 5g network the 5g technology is increasingly mentioned as a driving force of the fourth industrial revolution due to its characteristics and the possibilities it opens up. 5g is as important to humanity as was the invention of the steam engine in the first industrial revolution, the emergence of electricity and oil-powered devices in the second industrial revolution, and the emergence and spread of the internet in the third industrial revolution. the most important advancements that the 5g technology brings to communication are fast and reliable data transfers, extremely low latency, coverage, and energy efficiency required for such data transfer (10). the 5g technology improves communication in three main areas using the so-called “5g triangle” consisting of (11): • urllc: ultraseemedj 2021, vol 5, no. 1 telemedicine in otorhinolaryngology 125 southeastern european medical journal, 2021; 5(1) reliable low latency communication use cases; • mmtc: massive machine type communication (iot) use cases; • embb: enhanced mobile broadband – high-speed use cases. the technology called ultra-reliable low latency communications (urllc) brings improvements in the field of healthcare because it is extremely reliable in terms of communication continuity and has a very low response rate, which can be around five milliseconds or less (11). the latest example of a successful implementation of this technology was given by the istituto italiano di tecnologia in italy (12), where it was used on the body of a deceased person using robotic technology controlled via a 5g network. the procedure was performed in a manner that the surgeon effectively controlled the surgical robot, forceps, and laser and successfully performed a high-precision laser cordectomy as if he were physically present in the operating room. surgeries used to be limited due to unreliable and limited networks, which is a problem that the 5g network solves. in this case, it has been proven that a broader implementation of this technology is possible (12). in addition to surgery, the 5g network will enable fast and reliable transmission of the internet of things, which is a network of physical objects consisting of wireless sensors and portable minicomputers that collect data. such data will be stored in real time due to cloud computing, which provides computing services such as servers, storage, databases, networking, software, analytics, and intelligence (10). conditions for the application of telemedicine technology the biggest advantage of using telemedicine pertains to rural areas and areas of the world where there is a shortage of physicians (13,14). however, certain prerequisites are also required for telemedicine to be used. the application of telemedicine technology primarily depends on the development of telecommunication infrastructure and the relationship between institutions that provide specialist knowledge. at the moment, the most popular approach is based on the “hub-and-spoke” model, in which communications are established between an outpatient department (spoke) and specialist (hub) health care (15). the primary purpose of this model is to have a direct connection between an outpatient department and secondary health care in which there is direct communication between the patient, general practitioner and specialist. additionally, all sorts of online applications can also provide essential information about the patient and medical data. the fundamental principle is to gather all information we can about a patient in the spoke centre, which is then transferred to a hub centre specialist. this is the point where consultation begins. a primary health care centre must have medical equipment necessary to gather medical information and certain communication equipment, while specialists should only have multimedia equipment at their disposal. primary health care clinicians should have basic biometric instruments, such as thermometers, pressure gauges, oximeters, and ecg devices. only with these instruments can we gather important information required for monitoring the patients’ condition. besides biometrics, telemedicine uses all kinds of telemedicine applications that utilise advanced technology to provide static images and videos. using such interactive technology, we can ensure direct audio-visual communication and transfer static images, such as rtg, ct, or mr scans. it is precisely this technology that enables the classification of telemedicine into teleeducation, teleconsultation, telediagnosis, and telemonitoring (15). all of this is important because telemedicine provides telepresence, 3d visualisation and remote control supervision of patients, and better sharing of medical knowledge, which ultimately improves health care for patients. one of the main limitations of telemedicine in otorhinolaryngology and other medical fields is the need not only for the diagnosis of the disease, but also for treatment (16). therefore, for some cases when an examination is required, a portable haptic system that transmits a seemedj 2021, vol 5, no. 1 telemedicine in otorhinolaryngology 126 southeastern european medical journal, 2021; 5(1) palpatory impression has been developed, or ultrasound can be used (17,18). factors affecting the further development of telemedicine telemedicine has proved to be one of the most promising medical branches, with an ongoing increase in distribution worldwide (1). however, it still has not reached its full potential due to many significant reasons. one of the most critical problems regarding telemedicine distribution is the cost of telecommunication infrastructure and technology. sophisticated telemedicine devices are often over the budget of many hospitals all over the world, especially the ones from developing countries (1). secondly, to establish high-quality telemedicine communication with other medical centres worldwide, medical staff should undergo a proper training that will get the best out of the technology that will be used. it is essential to understand the complexity of medical staff involved in telemedicine projects – from physicians, nurses, and technicians to health managers, health administrators, and medical computer scientists. it is easy to conclude that providing these specific groups with training will also take time and funds. unfortunately, even when the highest standards in securing top-quality equipment and education for telemedicine are met, one cannot ignore a cultural factor in telemedicine distribution. many countries do not want to take advantage of modern medicine and are ignorant of creativeness, innovation, and diverse approaches to modern medical issues. if this factor changes over time, there will be significant advancements in modern medicine. other crucial aspects that most people do not take into account when discussing the factors affecting telemedicine distribution are legal boundaries, which differ from one country to another. for example, some of the united states of america’s legal boundaries restrict providing medical services outside of the country (1). therefore, it would not be legally possible to provide essential medical information to a specific country in europe that could help in a patient’s treatment outside the us. furthermore, distant telemedicine communication should not result in compromising and disclosing a patient’s private information, which is one of the most critical patient’s rights. because of the numerous advantages that modern telemedicine offers, the highest priority of every country should be to create stable and top-quality medical centres that provide telemedical services and to make them a reference centre. in order to achieve that, every country should have an assessment team, which would be in charge of the evaluation of certain medical centres in order to provide top medical services. unfortunately, most countries still have infrastructural and organisational limitations in that regard. even though there are various problems regarding telemedicine distribution, it is important to emphasise that telemedicine is growing increasingly in the entire world. with high motivation and acceptance of different ideas, telemedicine could be very successful in the future. special review of the use of telemedicine during the coronavirus pandemic covid-19 disease is a viral infection caused by the coronavirus named severe acute respiratory syndrome coronavirus 2 (sars-cov-2). the disease spread quickly across the globe. sarscov-2 transmission occurs via respiratory droplets produced by infected individuals while talking, sneezing, or coughing without covering the mouth and the nose (57). minimising the virus transmission has become a critical part of patient care during the covid‐19 pandemic. telemedicine is indeed advantageous in that regard as it prevents person-to-person contact, thus ensuring social distance, and it is therefore recommended by various health agencies for covid-19 (58). fortunately, telemedicine is not new to otorhinolaryngologists, so it can be applied in real-time delivery and monitoring of services via video conferencing (synchronous care). on the other hand, there is a store-andseemedj 2021, vol 5, no. 1 telemedicine in otorhinolaryngology 127 southeastern european medical journal, 2021; 5(1) forward methodology, where test results are stored and reviewed later (asynchronous care). these evaluations have been proven useful for plastic postoperative evaluation, head and neck oncology, facial trauma evaluation, triaging and postoperative visits, allergic or non-allergic rhinitis management, endoscopic sinus surgery, postoperative evaluation, and vocal rehabilitation. the covid‐19 pandemic has propelled telemedicine necessity and utilisation into the mainstays of current otorhinolaryngology practice (59). application in audiology audiologists have the opportunity to provide services to those who may otherwise have no access to much-needed care, from those in rural areas to patients with limited physical mobility (19). hearing aid services, such as counselling, fitting, and device testing can be provided through telemedicine. lack of high-speed internet connections and limited bandwidth in some rural locations could prevent the delivery of some services (20). telemedicine in audiology can be applied in real-time delivery and monitoring of services via videoconference. on the other hand, there is a store-and-forward methodology, where test results are stored and reviewed later. likewise, telemedicine in audiology has been applied in providing education and training to professionals, hearing screening, diagnosis, and intervention (21). small fitting of hearing aids is possible via the internet. the technological approach can help with hearing rehabilitation, as patients can ask for help from the comfort of their own homes (22). furthermore, teleaudiometry is a reliable, valid, and viable option for hearing tests. results can be analysed by experts remotely. moreover, teleaudiometry reduces the costs of hearing screening programs (23). telemedicine in audiology opens access to vital hearing care. telemedicine in phoniatrics and phonosurgery phoniatrics is a branch of otorhinolaryngology that studies, diagnoses, and deals with voice, speech, language, and swallowing disorders and diseases. the application of telemedicine in phoniatrics and phonosurgery is continuously growing and it can be an essential tool in certain medical situations, such as stuttering or dysphonia (1). the possibility of consulting with colleagues via web conference technology can sometimes be crucial in patient treatment options. according to some reports, around 12% of all speech therapies and assessments in india were performed via telemedicine (1)! the most important aspect of presenting a phoniatric patient via remote stroboscopy and laryngoscopy is high video and audio recording quality. higher quality of the footage will provide precise visual and auditory information about particular laryngeal pathology and facilitate physicians’ decision about treatment. furthermore, telemedicine can be essential in voice rehabilitation. it allows for direct consultations with experts across the world on certain phonological issues and provision of optimal healthcare to a patient by eliminating the barriers of time and space. a fascinating and successful example of telemedicine usage in phoniatrics is the camperdown program, a therapeutic approach in the treatment of stuttering (24-27). this therapeutic approach showed no significant difference in curing stuttering telematically in comparison to the in-person approach (28-31). this is only one of many examples highlighting telemedicine as one of the most critical and trustworthy approaches in modern phoniatrics. unfortunately, the cost of sophisticated technology and its complexity, which requires trained personnel, are often a limiting factor for a vast number of otorhinolaryngology departments. however, every medical professional should look forward to dealing with current barriers with a great deal of optimism and motivation to improve every aspect of telemedicine and make it available to every single part of the world. application in rhinology seemedj 2021, vol 5, no. 1 telemedicine in otorhinolaryngology 128 southeastern european medical journal, 2021; 5(1) numerous studies have proven that telemedicine consultation can be applied in rhinology as well. setzen et al. pointed out that it is possible to triage patients who need to undergo nasal endoscopy by taking a medical history through telemedicine, but this requires guidelines that need to be determined in the future (32). patients with unilateral sinonasal symptoms and patients with radiologically proven unilateral sinonasal diseases need repeated medical check-ups after nasal endoscopy via telemedicine. also, during the covid-19 pandemic, high-risk, immunocompromised patients present a challenge. telemedicine allows such patients to stay at home during their first examination. in immunocompromised patients, nasal endoscopy should be indicated at the onset of new symptoms, such as severe pain, fever, and other general symptoms (32). when it comes to ent, patients’ condition can be assessed by telemedicine by radiological images (x-ray, ct) and endoscopic images, which can be qualitatively transmitted to distant places. telemedicine technology has made rapid progress in the last 20 years in terms of the quality of equipment, better internet connectivity, and better image sharing software. in ent, synchronous telemedicine is also being developed, which has significant advantages over older methods (33,34). nasal endoscopy is a high-risk test due to a potential exposure of the examiner to a covid19 infection. also, anaesthetic sprays have aerosol potential, which increases the risk of infection transmission, both by direct contact and indirectly. as numerous studies have shown a correlation and concordance in the evaluation of sinus disorders between ct and nasal endoscopy, ct of the paranasal sinuses represents an interesting alternative to nasal endoscopy (35). another alternative is intranasal imaging, where, for instance, video files can be sent to a remote device (36). epistaxis is a common disorder in rhinology, which may be adaptable for a remote evaluation. telemedicine can help detect trigger risk factors and help cure minor bleeding. a history of uncontrolled bleeding, despite conservative measures provided, requires a prompt live evaluation. in rhinology, telemedicine may also play a role in a postoperative follow-up of patients after a nasal septal surgery and a functional endoscopic sinus surgery (32). telemedicine is being developed and is useful in skull base surgery. telementoring is widely applicable in skull base surgery, in endoscopic endonasal surgery for the surgical treatment of diagnoses such as benign and malignant neoplasms, cerebrospinal fluid leak, and inflammatory diseases. this technology requires two-way real-time video and audio communication using the existing technology. an expert who is not present in the operating room and a surgeon communicate using telemedicine technology and they have robotic technology available to visualise the operating field. also, with the help of a laser pointer, orientation can be assisted (37). application in head and neck oncology telemedicine is widely applicable in surgical head and neck oncology. in this area of otorhinolaryngology, the application of telemedicine results in better patient followups, better collaboration among physicians, reduction of unnecessary patient’s transfer to specialist facilities, and reduction of patient travel costs (38-41). kohlert et al. show that 48.6% of consultations in otorhinolaryngology account for head and neck oncological surgery (42). a study from the usa conducted by prasad et al. describes a virtual examination of patients with head and neck cancer, aiming to set out guidelines for each specific tumour site for the best follow-up possible (43). while extremely useful, this approach can be challenging for both the physician and the patient, mainly because it concerns a new clinical evaluation model for both parties. head and neck cancer surveillance is often challenging to perform in person, and doing this virtually is even more difficult (38,44). a multidisciplinary team participates in treating an oncology patient and communication and cooperation between team members are improved using real-time video conferencing (45-49). also, this form of communication seemedj 2021, vol 5, no. 1 telemedicine in otorhinolaryngology 129 southeastern european medical journal, 2021; 5(1) between multidisciplinary team members has been applied during the covid-19 virus pandemic due to the inability to gather team members in one place. in addition to real-time video conferencing, communication can be established using various communication platforms. rimmer et al. stated that for some patients, postoperative telemedicine monitoring is relatively safe and saves time (48). also, during the covid-19 epidemic at our institution, psychological counselling was provided to oncology patients by phone, which according to some studies had a positive effect on this type of patients (50,51). application in palliative care less than a decade ago, the worldwide palliative care alliance and the world health organization concluded that palliative health care represents both a necessity and an essential human right (52). however, palliative care has been suffering from a significant lack of resources for years, primarily in terms of finances. the situation is further aggravated by the ongoing pandemic. some researchers have noticed that head and neck tumours have shown some similarities with the covid-19 infection. regarding both conditions, resources are low, there is a shortage of time, patients’ condition can quickly deteriorate, and the family needs consulting about these diagnoses (52). therefore, the majority of head and neck tumour centres have allowed consultations with patients and their families by phone. the most important goal is to lower the number of unnecessary hospitalisations and consequently reduce the risk of a coronavirus infection for those patients. another equally important thing is to provide secondary health care through a “hub-spoke” model, in which human empathy and a professional relationship between primary and secondary care physicians should play the key role (52). we live in the age of technology and one should make use of its resources because the definition of palliative care is to provide the best health care possible and to improve the quality of life of patients suffering from a disease in its terminal stage. this is where telemedicine comes in with its potential to improve health care to a degree which every terminal head and neck oncology patient deserves. therefore, it is necessary to work with telemedicine technology on further development of palliative care, and in otorhinolaryngology, this primarily applies to patients suffering from head and neck cancers (53,54). application in sleep medicine obstructive sleep apnoea (osa) is a common disease that leads to severe symptoms and comorbidities. although general measures are essential, continuous positive airway pressure (cpap) is the best treatment option (55). however, compliance may be suboptimal and telemedicine may play a role in improving it. to improve osa management, there is an urgent need to develop new cost-effective strategies, especially those related to osa treatment, from lifestyle changes to cpap implementation. two broad strategies should be applied: 1) appropriate pre-, peri-, and post-titration measures to ensure sufficient continuous positive airway pressure, appropriate training, and appropriate support during follow-up; and 2) use of technological advances, including the optimisation of cpap devices and the use of telemedicine, specifically focused on the first days or weeks of treatment (55,56). telemedicine can help with these processes, especially when tailored to each patient. traditional methods of diagnosis and treatment of osa include history, clinical examination, diagnosis, counselling on the condition and treatment of the patient, patient care, and selftreatment of patients with mild or severe obstructive sleep apnoea (56). a multidisciplinary approach is required for an optimal diagnosis and treatment of patients with osa. the neurologist, otorhinolaryngologist, and head and neck surgeon play the most important role in this multidisciplinary team. the neurologist diagnoses obstructive sleep apnoea by using polysomnography, the golden diagnostic standard. the otorhinolaryngologist tries to locate the site of airway obstruction in a patient by performing a standard clinical seemedj 2021, vol 5, no. 1 telemedicine in otorhinolaryngology 130 southeastern european medical journal, 2021; 5(1) examination, including awake fibre endoscopy and drug-induced sleep endoscopy (dise). the head and neck surgeon’s role is surgical treatment of the detected obstruction and consequent improvement of airflow through the upper respiratory tract. adhering to the guidelines and best practices of treatment and follow-up of patients, there are also numerous opportunities for improvement with new technologies. telemedicine can improve the treatment and follow-up of patients with obstructive sleep apnoea by educating patients more efficiently and reducing the costs of treatment (56). medical history is essential for a correct diagnosis, and it can be done by phone, video link, or e-mail consultations. completion of surveys can also take place electronically. measurements of body weight, bmi, height, and blood pressure can be taken at home, and the results can be sent electronically. replacing the examination of patients, such as a specialist examination by an otorhinolaryngologist, with video call examinations is challenging. this is because the examination involves methods such as indirect laryngoscopy, fibre endoscopy, or dise, so in this case, one-on-one examination is essential. monitoring treatment outcomes and the condition of patients with obstructive sleep apnoea by other specialists can be facilitated by telemedicine because the cpap device can send information about the patient and therapy given (55). as with other diseases and conditions, osa telemedicine plays a vital role in the communication between members of a multidisciplinary team, who can share experiences, educate themselves, monitor the patient, and decide on further treatment through videoconferencing. conclusion the development of telemedicine in otorhinolaryngology represents a new approach to diagnostic and therapeutic procedures. this is a rapidly evolving field, which certainly introduces some novelties into everyday practice, improves the quality and availability of medical care in areas where there is a shortage of physicians, and increases the attractiveness of otorhinolaryngology to younger colleagues. it is also necessary to point out the shortcomings of telemedicine technology – primarily expensive equipment and the need for additional staff training. in countries such as croatia, where there is no shortage of otorhinolaryngologists and resources are limited, telemedicine can be beneficial in situations such as the covid-19 pandemic and it can also be used for educational purposes. acknowledgement. none.. disclosure funding. no specific funding was received for this study. transparency declaration: competing interests: none to declare references 1. beule ag. telemedical methods in otorhinolaryngology. laryngorhinootologie. 2019; 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197(1):117-26. https://doi.org/10.1164/rccm.201703-0582oc 56. schutte-rodin s. telehealth, telemedicine, and obstructive sleep apnea. sleep med clin. 2020; 15(3):359-75. https://doi.org/10.1016/j.jsmc.2020.05.003 57. machhi j, herskovitz j, senan am, dutta d, nath b, oleynikov md, blomberg wr, meigs dd, hasan m, patel m, kline p, chang rc, chang l, gendelman he, kevadiya bd. the natural history, pathobiology, and clinical seemedj 2021, vol 5, no. 1 telemedicine in otorhinolaryngology 134 southeastern european medical journal, 2021; 5(1) manifestations of sars-cov-2 infections. j neuroimmune pharmacol. 2020; 15(3):359-86. https://doi.org/10.1007/s11481-020-09944-5 58. laskar p, yallapu mm, chauhan sc. "tomorrow never dies": recent advances in diagnosis, treatment, and prevention modalities against coronavirus (covid-19) amid controversies. diseases. 2020; 8(3):30. https://doi.org/10.3390/diseases8030030 59. singh ak, kasle da, jiang r, sukys j, savoca el, z lerner m, kohli n. a review of telemedicine applications in otorhinolaryngology: considerations during the coronavirus disease of 2019 pandemic. laryngoscope. 2020: 10.1002/lary.29131. https://doi.org/10.1002/lary.29131 1 author contribution. acquisition of data: mihalj h, zubčić ž, včeva a, vranješ ž, maleš j, birtić d, mendeš t, milanković sg, prpić t, bogović v, abičić i, rezo m, moguš m, šestak a administrative, mihalj h, zubčić ž, včeva a, vranješ ž, maleš j, birtić d, mendeš t, milanković sg, prpić t, bogović v, abičić i, rezo m, moguš m, šestak a analysis and interpretation of data: mihalj h, zubčić ž, včeva a, vranješ ž, maleš j, birtić d, mendeš t, milanković sg, prpić t, bogović v, abičić i, rezo m, moguš m, šestak a conception and design: mihalj h, zubčić ž, včeva a, vranješ ž, maleš j, birtić d, mendeš t, milanković sg, prpić t, bogović v, abičić i, rezo m, moguš m, šestak a critical revision of the article for important intellectual content: mihalj h, zubčić ž, včeva a, vranješ ž, maleš j, birtić d, mendeš t, milanković sg, prpić t, bogović v, abičić i, rezo m, moguš m, šestak a drafting of the article: mihalj h, zubčić ž, včeva a, vranješ ž, maleš j, birtić d, mendeš t, milanković sg, prpić t, bogović v, abičić i, rezo m, moguš m, šestak a final approval of the article: mihalj h, zubčić ž, včeva a, vranješ ž, maleš j, birtić d, mendeš t, milanković sg, prpić t, bogović v, abičić i, rezo m, moguš m, šestak a guarantor of the study: mihalj h, zubčić ž, včeva a, vranješ ž, maleš j, birtić d, mendeš t, milanković sg, prpić t, bogović v, abičić i, rezo m, moguš m, šestak a provision of study materials or patients: mihalj h, zubčić ž, včeva a, vranješ ž, maleš j, birtić d, mendeš t, milanković sg, prpić t, bogović v, abičić i, rezo m, moguš m, šestak a statistical expertise (statistical analysis of data): mihalj h, zubčić ž, včeva a, vranješ ž, maleš j, birtić d, mendeš t, milanković sg, prpić t, bogović v, abičić i, rezo m, moguš m, šestak a seemedj 2020, vol 4, no. 1 employment after liver transplantation 49 southeastern european medical journal, 2020; 4(1) original article employment of patients after liver transplantation 1 anita holetić 1, mirjana đukić 2, lada zibar* 3, 4 1 department of surgery, merkur university hospital, zagreb, croatia 2 health centre zagreb – centre, zagreb, croatia 3 department of nephrology, merkur university hospital, zagreb, croatia 4 faculty of medicine, josip juraj strossmayer university of osijek, croatia *corresponding author: lada zibar, ladazibar @gmail.com received: feb 13, 2020; revised version accepted: mar 22, 2020; published: apr 27, 2020 keywords: liver transplantation, employment, liver disease, health-related quality of life abstract aim: to determine the prevalence of employment of patients after liver transplantation (tx) and the history of employment, to compare employment with patients’ opinions about their ability to work and to establish possible reasons for frequent unemployment. methods: ninety-eight respondents participated in the study. they were the first 98 liver transplant patients who came for a check-up at merkur university hospital by the time of the research and agreed to participate in the study. we created and used a questionnaire about the level of education and employment prior to and after the liver tx. results: before the diagnosis of liver disease, 59.18 % of the patients were employed, while after liver tx, at the time of the research, the employment rate decreased to 8.2 %. during the same time span, the number of retired patients increased from 3.1 % before the diagnosis to 63.3 % after liver tx at the time of the research. the main reasons for unemployment were poor health due to liver disease and employers’ unwillingness to hire these patients because of a potential risk of adjustment of working hours. median follow-up time after liver tx was 3 years (interquartile range 2 – 6). conclusion: many patients with severe liver disease are unemployed. liver tx did not increase the rate of employment of croatian patients. patients should be supported by society in finding appropriate employment. (holetić a, đukić m, zibar* l. employment of patients after liver transplantation. seemedj 2020; 4(1); 49-54) seemedj 2020, vol 4, no. 1 employment after liver transplantation 50 southeastern european medical journal, 2020; 4(1) introduction liver transplantation (tx) is the standard method of treatment of end-stage liver disease (esld), acute liver failure and selected cases of hepatocellular carcinoma. patients who underwent tx have better survival rates. due to an improvement in the surgical treatment and modern immunosuppressive medicaments, the 5-year survival rate increased up to 70 % (1,2,3). the term “health-related qol” (hrqol, healthrelated quality of life) has been in use for several decades. this term encompasses the effect of health, i.e. of the illness and of treatment of the illness on the patient’s physical, cognitive and social functioning. ability to work is considered to be a significant indicator of well-being and health status (4,5). liver tx, as the best method of treatment of esld, improves survival, health status, qol and ability to work (6-8). however, transplant patients frequently face unemployment, sick leave and retirement. we thus presumed that liver tx, although the best method for the treatment, unfortunately does not improve the patients’ access to employment. the aims of the study were to determine the prevalence of employment of liver transplant patients, to compare employment with the patients’ opinions about their ability to work and to establish possible reasons for frequent unemployment. there are no available published data about the employment of liver transplant patients in croatia to date. patients and methods the research was conducted at merkur university hospital in zagreb in 2018. ninetyeight liver transplant patients were selected as the subjects of our study. the first 98 patients who came for a check-up by the time of the research were included in the study. the patients were asked to fill in a questionnaire. the questionnaire was created specifically for our study by the researcher and it included data about employment before the diagnosis of liver disease, before and after liver tx and at the time of the research. we separately compared overall employment and working patients. overall employment included all employed participants, even those who were on sick leave. demographic data and data about liver disease were taken from medical records. seventy (71.4 %) participants were male and 28 (28.6 %) were female. participants were divided into groups according to age, with an interval of 10 years. fifteen participants (15.3 %) were born in the period between 1941 and 1950. most participants, 42 (42.9 %) of them, were born between 1951 and 1960, while 33 (33.7 %) participants were born between 1961 and 1970. the median age of participants was between 58 and 67 years (interquartile range, iqr, 48 – 67). the youngest patient was 26 years old and the oldest one was 67 years old (figure 1). median follow-up time (from liver tx to the research) was 3 years (iqr 2 – 6). figure 1. respondents divided into groups according to age, with an interval of 10 years (n = 98) 15 42 33 4 3 1 0 5 10 15 20 25 30 35 40 45 1941-1950 1951-1960 1961-1970 1971-1980 1981-1990 1991-2000 n birth year seemedj 2020, vol 4, no. 1 employment after liver transplantation 51 southeastern european medical journal, 2020; 4(1) statistical analysis data were statistically analysed using spss (version 16.0. spss inc., chicago, il, usa). descriptive statistics included the median with iqr for numeric data. absolute and relative frequencies were used for nominal data. differences were obtained by the chi-square test. statistical significance was accepted if p was < 0.05. results ninety-eight patients were included in the research. of 98 participants, 19 completed only primary education or less (19.4 %), 58 finished secondary education (59.2 %), 12 had higher education qualifications (12.2 %) and 9 had a university degree (9.2 %). most of the participants, 50 of them (51 %), waited for liver tx for more than a year. twenty-nine participants (29.6 %) waited for liver tx less than six months, while 19 of them (19.4 %) waited between 6 months and a year. all the patients selected for the research underwent liver tx between 2002 and 2017. at the time of the research (in 2018), 4 participants already had two liver txs. most of the participants, 21 of them (21.4 %), underwent liver tx in 2017. in 2016, 17 participants underwent liver tx, while 14 of them had liver tx in 2015. compared to the period before receiving the diagnosis of severe liver disease, employment among the working patients before liver tx decreased by 37.8 % (59.2 % before the diagnosis, 21.4 % before liver tx). overall employment decreased by 13.2 % (62.2 % before the diagnosis, 48.9 % thereafter). the number of patients on sick leave (for more than a month) increased by 9 times (3 before the diagnosis, 27 just before tx) (figure 2).. figure 2. employment of patients (n = 98) before diagnosis of liver disease and just before liver transplantation (tx) the number of patients who were working early after tx decreased by 9.2 % compared to the period just before tx (21.4 % just before tx, 12.2 % early after tx). overall employment decreased by 10.2 % (49 % just before tx, 38.8 % early after tx) (figure 3). . 57 1 3 9 27 1 19 2 27 14 35 1 0 10 20 30 40 50 60 working full-time working part-time sick leave unemployed retirement student n before diagnosis of liver disease before tx seemedj 2020, vol 4, no. 1 employment after liver transplantation 52 southeastern european medical journal, 2020; 4(1) figure 3. employment of patients (n = 98) just before and early after liver transplantation (tx) at the time of the research, the number of employed patients decreased by 4 % compared to the period early after tx (12.2 % early after tx, 8.2 % at the time of the research). the number of patients using sick leave decreased by 7.1 % in the same period (26.5 % early after tx, 19.4 % at the time of research) (figure 4). figure 4. employment of patients (n = 98) early after liver transplantation (tx) and at the time of the research at the time of the research, the number of employed patients decreased by 51 % compared to the period before the diagnosis of esld (59.2 % before the diagnosis, 8.2 % at the time of research). in the same period, the number of 19 2 27 14 35 1 11 1 26 9 50 1 0 10 20 30 40 50 60 working full-time working part-time sick leave unemployed retirement student n before tx after tx p = 0.285 11 1 26 9 50 1 7 1 19 9 62 0 0 10 20 30 40 50 60 70 working full-time working part-time sick leave unemployed retirement student n after tx at the time of research seemedj 2020, vol 4, no. 1 employment after liver transplantation 53 southeastern european medical journal, 2020; 4(1) patients on sick leave increased by 16.3 % (3.1 % before the diagnosis, 19.4 % at the time of the research) and the number of retired patients increased by 60.2 % (3.1 % before the diagnosis, 63.3 % at the time of the research) (figure 5). figure 5. employment of patients (n = 98) before the diagnosis of liver disease and at the time of the research at the time of the research, 8 participants were employed (8.2 %), while 34 felt capable of working (34.7 %). nineteen participants felt capable of working full-time, while 15 felt capable of working part-time (p = 0.773). as the main reason for unemployment, 22 patients (22.5 %) reported poor health due to esld, 4 of them (4.1 %) claimed that employers did not want to hire workers who underwent liver tx, 22 of them reported some other reason, while 41 did not give an answer. discussion this is the first research on employment of liver tx patients in croatia. just before liver tx, overall employment had already decreased by 13.2 % compared to the period before receiving the diagnosis of severe liver disease. although patients presumably have a better health status and qol after liver tx, the number of employed patients early after liver tx decreased by 9.2 % compared to the period just before liver tx. at the time of the research, only 8.2 % of patients were employed. many researchers described similar results regarding employment after liver tx. in 2018, e. r. waclawski and p. noone published a systematic review of the impact of liver tx on employment (9). they included 13 studies published from january 2001 to december 2016. all the studies they found showed either a fall in the employment rate or no effect on the employment rate. overall employment ranges from 18 to 44 % after liver tx, which was higher than in our research. employment after liver tx was lower than after other organ tx, such as kidney or heart tx, but higher than after lung tx, as shown in a study which included 281 kidney, heart, liver and lung transplant patients (10). compared to the research about employment after kidney tx in croatia, conducted at the clinical hospital centre osijek in 2017, employment after liver tx at the time of the research was lower than after kidney tx (namely 18.1 %), which corresponded to the results mentioned earlier (11). what was the reason of unemployment after tx? after tx, patients need to take immunosuppressive medicaments for the rest of their lives. immunosuppressive therapy has adverse effects, including infections, diabetes mellitus, nephrotoxicity, cardiovascular disease and osteoporosis (12). moreover, patients have a mental burden because of the disease. however, our study has shown that 8.2 % of the 57 1 3 9 27 1 7 1 19 9 62 0 0 10 20 30 40 50 60 70 working full-time working part-time sick leave unemployed retirement student n before diagnosis of liver disease at the time of research p = 0.773 seemedj 2020, vol 4, no. 1 employment after liver transplantation 54 southeastern european medical journal, 2020; 4(1) participants were employed, while 34.7 % of them felt capable of working, which indicates a significant disproportion. the question is what caused that disproportion. as the main reason for unemployment, the majority of respondents reported poor health due to esld, while the minority of respondents claimed that employers did not want to hire the patients who underwent tx. conclusion even though tx improves the patients’ health status and survival, it is necessary to ensure that they have a better qol and allow them to return to their usual activities, including appropriate employment. social support is needed to improve the chances of employment. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. . references 1. masala d, mannocci a, unim b, del cimmuto a, turchetta f, gatto g, santoro r, ettorre gm, boccia a, la torre g. quality of life and physical activity in liver transplantation patients: results of a case-control study in italy. transplant proc. 2012; 44:1346–1350. 2. lankarani kb, eshraghian k, malekhosseini sa, janghorban p, geramizadeh b, eshraghian a. outcomes of liver transplantation for patients with acute liver failure. arch iran med. 2013; 16:64–67. 3. adam r, mcmaster p, o'grady jg, castaing d, klempnauer jl, jamieson n, neuhaus p, lerut j, salizzoni m, pollard s, muhlbacher f, rogiers x, garcia valdecasas jc, berenguer j, jaeck d, moreno gonzalez e; european liver transplant association. evolution of liver transplantation in europe: report of the european liver transplant registry. liver transplantation. 2003; 9(12):1231– 43. 4. tavakoli-fard n, mortazavi s-a, kuhpayeh zadeh j, nojomi m. quality of life, work ability and other important indicators of women’s occupational health. international journal of occupational medicine and environmental health. 2015; 29(1):77–84. 5. chen p-x. health-related quality of life of 256 recipients after liver transplantation. world journal of gastroenterology. 2012; 18(36):5114. 6. onghena l, develtere w, poppe c, geerts a, troisi r, vanlander a, berrevoet f, rogiers x, van vlierberghe h, verhelst x. quality of life after liver transplantation: state of the art. world journal of hepatology. 2016;8(18):749. 7. butt z, parikh nd, skaro ai, ladner d, cella d. quality of life, risk assessment, and safety research in liver transplantation. current opinion in organ transplantation. 2012; 17(3):241– 7. 8. saab s, wiese c, ibrahim ab, peralta l, durazo f, han s, yersiz h, farmer dg, ghobrial rm, goldstein li, tong mj, busuttil rw. employment and quality of life in liver transplant recipients. liver transplantation. 2007; 13(9):1330–8. 9. waclawski er, noone p. systematic review: impact of liver transplantation on employment. occupational medicine. 2018; 68(2):88–95. 10. de baere c, delva d, kloeck a, remans k, vanrenterghem y, verleden g, vanhaecke j, nevens f, dobbels f. return to work and social participation: does type of organ transplantation matter? transplantation. 2010; 89(8):1009–15. 11. zibar l, đukić m. employment in patients with renal replacement therapy. southeastern european medical journal. 2019; 3(1):11-20. 12. lim kbl, schiano td. long-term outcome after liver transplantation. mount sinai journal of medicine: a journal of translational and personalized medicine. 2012; 79(2):169–89. seemedj 2021, vol 5, no. 2 serum ferritin levels, arterial hypertension and shift work in women 27 southeastern european medical journal, 2021; 5(2) original article relationship between serum ferritin levels, arterial hypertension and shift work in women. a cross-sectional analysis 1 katica cvitkušić lukenda *1,2, domagoj vučić 1,2, antonija raguž 1, ivan bitunjac 1, domagoj mišković 1, krešimir gabaldo 1, blaženka miškić 1,3, marijana knežević praveček 1,3 1 department of internal medicine, “dr. josip benčević” general hospital, slavonski brod, croatia 2 university postgraduate interdisciplinary doctoral study programme in molecular biosciences, josip juraj strossmayer university of osijek, osijek, croatia 3 faculty of dental medicine and health, josip juraj strossmayer university of osijek, osijek, croatia *corresponding author: katica cvitkušić lukenda, kcvitkusiclukenda@gmail.com received: aug 12, 2021; revised version accepted: oct 21, 2021; published: nov 26, 2021 keywords: arterial hypertension, c-reactive protein, ferritin, shift work, women abstract introduction: the aim of this study was to use cross-sectional research to determine the relationship between serum ferritin levels and arterial hypertension in women who work in shifts and women with regular daytime working hours. methods: the respondents included 67 female nurses divided into two groups: nurses working in 12hour shifts (7 am to 7 pm/7 pm to 7 am) were the test group, while nurses regularly working for 8 hours (7 am to 3 pm) were the control group. data collection included information on associated diseases, chronic medication, last menstruation, duration of menopause, cigarette smoking, number of years of employment in shift work and regular daytime work, laboratory and anthropometric parameters and blood pressure levels. results: in all respondents, there was a significant and positive relationship between ferritin and crp levels, i.e. the higher the crp levels, the higher the ferritin levels (rho = 0.401; p = 0.001). among respondents who have regular daytime working hours, there was no significant association between ferritin and other indicators, while in the group of those who work in shifts, there was a significant and positive association between ferritin and crp (rho = 0.468; p = 0.002). finally, a positive correlation was found between the number of years of employment in shift work and systolic blood pressure levels, i.e. higher systolic pressure was observed in those respondents who worked longer in shifts (rho = 0.424, p = 0.03). conclusion: the study demonstrated a significant correlation between the number of years of employment in shift work and systolic blood pressure. a positive correlation between serum crp and ferritin levels was also observed in all respondents, and especially in shift workers. (cvitkušić lukenda k, vučić d, raguž a, bitunjac i, mišković d, gabaldo k, miškić b, knežević praveček m. relationship between serum ferritin levels, arterial hypertension and shift work in women. a cross-sectional analysis. seemedj 2021; 5(2); 27-37) seemedj 2021, vol 5, no. 2 serum ferritin levels, arterial hypertension and shift work in women 28 southeastern european medical journal, 2021; 5(2) introduction iron is a trace element that is necessary for the normal functioning of numerous metabolic processes in living beings [1]. accumulation of iron in organs such as the liver, heart and endocrine glands can lead to pathological changes and dysfunction of the organs [2]. ferritin is an intracellular protein that serves to store and release iron, and by determining its serum level, insights into the body’s iron stores are obtained [3, 4, 5]. in addition to storing and releasing iron, ferritin plays a role in cell proliferation, angiogenesis, immunosuppression and atherosclerosis [6]. atherosclerotic lesions have been shown to contain ferritin; this means that iron is a potent catalyst for oxidation of lipids found in ldl cholesterol, which plays a major role in atherogenesis [7, 8]. it is known that the presence of ferritin is a result of the inflammatory response of the acute phase of systemic infections, because its synthesis is increased under the influence of cytokines and its serum level increases in inflammatory events [9]. observational studies have shown a positive correlation between serum ferritin levels and the development of chronic diseases such as atherosclerotic coronary artery disease and cancers, while certain other studies have failed to show a correlation between high serum ferritin levels and chronic diseases [10, 11]. arterial hypertension is one of the leading causes of morbidity and mortality in the population, with a continuous correlation between blood pressure levels and cardiorenal events [12]. previous studies have found a higher prevalence of arterial hypertension in men who had higher iron stores and serum ferritin levels [13, 14, 15]. likewise, a positive correlation has been shown between serum ferritin levels and the prevalence of arterial hypertension in shift workers [16]. compared to regular daytime (morning and afternoon) work, shift work (day and night shifts) leads to circadian rhythm disorders and, additionally due to lifestyle changes, to an increase in stress levels, which can trigger an inflammatory response. such an inflammatory response has an important role in all stages of atherosclerotic plaque formation and development [17]. previous research has confirmed the association between increased arterial stiffness and the development of atherosclerotic plaque [18, 19]. a positive correlation between serum ferritin levels and arterial stiffness in adult men and women has also been confirmed [20]. unlike in men, serum ferritin levels in women change before and after menopause. during the reproductive period, serum ferritin levels in women are low due to menstrual bleeding, but in menopause, after menstrual cycles stop, serum ferritin levels increase. natural postmenopause is defined by the absence of menstrual bleeding over a period of 12 months (excluding exogenous factors such as hysterectomy) [21]. in addition, serum ferritin levels in women of childbearing age are difficult to assess because throughout life, a number of factors can affect the levels, such as pregnancy, hormone therapy and gynaecological diseases [22]. consequently, the association between serum ferritin levels and arterial hypertension in women has been insufficiently investigated. to our knowledge, there are no studies concerning the association between serum ferritin levels, arterial hypertension and shift work in women. therefore, the aim of this article was to use cross-sectional research to determine the relationship between serum ferritin levels and arterial hypertension in women who work in shifts and those with regular daytime working hours. respondents and methods respondents of the total number of healthcare professionals who underwent a health check-up at the “dr. josip benčević” general hospital in slavonski brod, croatia, in the period from may to july 2021, 67 female nurses were included in this study. the respondents were female nurses who were divided into two groups: nurses working in 12hour shifts (7 am to 7 pm/7 pm to 7 am) were the seemedj 2021, vol 5, no. 2 serum ferritin levels, arterial hypertension and shift work in women 29 southeastern european medical journal, 2021; 5(2) test group, while nurses regularly working for 8 hours (7 am to 3 pm) were the control group. the inclusion criteria were female gender, age of respondents between 18 and 65 for both groups, consent to participation in the survey and signed informed consent. exclusion criteria included any form of iron replacement therapy, malignant disease, chronic renal failure, chronic liver disease, severe anaemia, acute illness, hormone replacement therapy, immunomodulatory therapy, reluctance to participate in the research. the study was conducted in accordance with the declaration of helsinki. all participants in the survey signed a written informed consent form. the research was approved by the ethics committee of the “dr. josip benčević” general hospital in slavonski brod (ethical approval no. 04000000/21-46). data collection the questionnaire was given and the physical examination was performed as part of the annual internal medicine examination. the questionnaire consisted of a report on medical history and primary health: it included data on chronic medication (for chronic conditions such as arterial hypertension, dyslipidaemia, diabetes, chronic obstructive pulmonary disease, osteoarthritis, depression), last menstruation, duration of menopause, cigarette smoking, number of years of employment in shift work and regular daytime work (at least one year for both groups). regular cycles were the marker of premenopausal women, while absence of menstruation for the last 12 months or longer was a criterion for postmenopausal women [21]. cigarette smoking habits were recorded as smoker, ex-smoker and non-smoker. blood pressure was measured after 5 minutes of rest. three measurements were performed at 1minute intervals, with the respondents in a sitting position and using their right arm. omron m6 comfort® blood pressure monitor and omron hem-fl31® upper arm cuff with circumference of 22–42 cm were used to measure blood pressure. levels of systolic pressure ≥ 140 mmhg and diastolic pressure ≥ 90 mmhg were used to define arterial hypertension, in accordance with the current guidelines for arterial hypertension of the european society of cardiology [12]. the blood pressure level of the respondents was calculated as the mean value of the second and third measurements. respondents who had previously taken antihypertensive therapy were placed in the group with proven arterial hypertension. height and weight were measured to the nearest 0.1 cm and 0.1 kg (using the seca® scale and altimeter). during the measurement, the respondents were asked to take off their clothes and put on a disposable dress for the examination. waist circumference (wc) was measured to the nearest 0.1 cm at the midpoint between the lower costal arch and the iliac crest. body mass index (bmi) was calculated as weight/height ratio2 (kg/m2); values ≥ 25 kg/m2 indicated that the respondent is overweight, while values ≥ 30 kg/m2 indicated obesity. serum ferritin concentration was measured using the alinity® immunochemical analyser (abbott). laboratory parameters tested using fasting blood samples were as follows: erythrocytes (e), haemoglobin (hb), haematocrit (htc), mean corpuscular volume (mcv), leukocytes (l), platelets (tr), iron (fe), unsaturated iron-binding capacity (uibc), total iron-binding capacity (tibc), total cholesterol (tc), low-density lipoprotein (ldl), high-density lipoprotein (hdl), triglycerides (tg), c-reactive protein (crp), blood glucose (bg), haemoglobin a1c (hba1c) and vitamin d. complete blood count was determined using the sysmex xn1000® haematology analyser by fluorescence flow cytometry. vitamin d levels were determined by chemiluminescent immunochemical assay using the alinity® immunochemical analyser (abbott). haemoglobin a1c in venous blood was determined using the dxc 700 au® biochemical analyser. finally, biochemical parameters crp, bg, tc, tg, hdl, ldl, fe and uibc were determined using the biochemical part of the integrated alinity® system (abbott). seemedj 2021, vol 5, no. 2 serum ferritin levels, arterial hypertension and shift work in women 30 southeastern european medical journal, 2021; 5(2) statistical analysis general characteristics of the respondents, according to the groups (shift work/regular daytime work), were derived using a descriptive method for continuous variables and using the chi-squared test for categorical variables after data weighting. the data were sorted into quartiles based on the respondents’ serum ferritin concentrations: quartile 1 (q1) ≤ 26.11 ng/ml, quartile 2 (q2) 26.12–47.74 ng/ml, quartile 3 (q3) 47.75–93.5 ng/ml, and quartile 4 (q4) > 93.5 ng/ml. the respondents’ basic characteristics according to ferritin levels in the quartiles were derived using the chi-squared test and fisher’s exact test. by dividing data into quartiles, four groups were obtained with an almost equal number of respondents. appropriate nonparametric tests were used to compare two or more independent groups. the mann–whitney u test was used to compare age, bmi, number of years of work and blood pressure levels between the test and control groups. the kruskal–wallis test (conover posthoc) was used to compare age, bmi, number of years of work, blood pressure levels in relation to groups, according to ferritin levels in the quartiles. the chi-squared test was performed to divide the respondents according to ferritin levels and the presence of arterial hypertension. spearman’s correlation coefficient was used to assess the association of ferritin and other biochemical values between groups and to assess the association of shift work with arterial pressure and ferritin levels. the level of statistical significance was set at p < 0.05. statistical analysis was performed using the spss for windows 11.0.3 software (spss inc., chicago, il, usa). results the research was conducted on 67 respondents, of whom 43 (64%) work in shifts and 24 (36%) regularly work 8 hours. there were 45 (67.2%) obese or overweight respondents. there were 42 (62.7%) respondents in postmenopause, and in regard to comorbidities, arterial hypertension was recorded in 22 (32.8%) respondents, while diabetes was present in 3 of them (4.5%). out of a total of 51 respondents who gave an answer about smoking, 19 (62.7%) respondents smoked cigarettes daily. antihypertensive therapy was taken by 19 (37.3%), statins by 6 (9%), and vitamin d by 3 (4.5%) respondents. there were no significant differences in general characteristics between the groups (table 1). table 1. basic characteristics of the respondents number (%) of respondents p* regular daytime work (n = 24) shift work (n = 43) total bmi kg/m2 < 25, normal 7 (29.2) 15 (34.9) 22 (32.8) 0.71 25–30, overweight 12 (50) 17 (39.5) 29 (43.3) > 30, obese 5 (20.8) 11 (25.6) 16 (23.9) postmenopause 14 (58.3) 28 (65.1) 42 (62.7) 0.58 smoking 6 (28.6) 13 (43.3) 19 (37.3) 0.28 arterial hypertension 11 (45.8) 11 (25.6) 22 (32.8) 0.09 diabetes mellitus 2 (8.3) 1 (2.3) 3 (4.5) 0.25 antihypertensive therapy 10 (41.7) 9 (20.9) 19 (37.3) 0.07 statins therapy 4 (16.7) 2 (4.7) 6 (9) 0.09 vitamin d therapy 1 (4.2) 2 (4.7) 3 (4.5) 0.93 *2 test seemedj 2021, vol 5, no. 2 serum ferritin levels, arterial hypertension and shift work in women 31 southeastern european medical journal, 2021; 5(2) a statistically significant interquartile difference was observed in postmenopausal respondents (table 2).. table 2. basic characteristics of the respondents according to ferritin levels number (%) of respondents according to ferritin levels p* < 26.11 (n = 17) 26.12–47.74 (n = 17) 47.75–93.5 (n = 17) > 93.5 (n = 16) total q1 q2 q3 q4 bmi kg/m2 < 25, normal 8 (47.1) 7 (41.2) 4 (23.5) 3 (18.8) 22 (32.8) 0.61 25–30, overweight 6 (35.3) 6 (35.3) 8 (47.1) 9 (56.3) 29 (43.3) > 30, obese 3 (17.6) 4 (23.5) 5 (29.4) 4 (25) 16 (23.9) postmenopause 4 (23.5) 11 (64.7) 13 (76.5) 14 (87.5) 42 (62.7) 0.001 smoking 5 (38.5) 5 (31.3) 5 (50) 4 (33.3) 19 (37.3) 0.79 arterial hypertension 2 (11.8) 7 (41.2) 7 (41.2) 6 (37.5) 22 (32.8) 0.20 diabetes mellitus 1 (5.9) 1 (5.9) 1 (5.9) 0 3 (4.5) > 0.99† antihypertensive therapy 1 (5.9) 7 (41.2) 5 (29.4) 6 (37.5) 19 (28.4) 0.08† statins therapy 0 2 (11.8) 2 (11.8) 2 (12.5) 6 (9) 0.56† vitamin d therapy 0 0 2 (11.8) 1 (6.3) 3 (4.5) 0.32† * 2 test; † fisher’s exact test the median age of the respondents was 57 (interquartile range of 45 to 60 years), with respondents ranging from 25 to 65 years old and no significant difference between the groups. there were no significant differences in bmi, wc, postmenopausal duration, systolic and diastolic pressure and number of years of treatment for arterial hypertension with respect to the groups. nurses working in shifts had more work experience, compared to nurses working only in the morning shift, with a median of 35 years (interquartile range from 25 to 39 years) (mann–whitney u test, p < 0.001) (tables 3 and 4). table 3. age, bmi, years of experience, blood pressure levels in relation to groups median (interquartile range) difference * 95% ci p† regular daytime work (n = 24) shift work (n = 43) age (years) 56.5 (44.5–60.75) 57 (45–59) 0 -4–4 0.98 bmi (kg/m2) 26.62 (24.33–29.43) 27.1 (23.9–30.1) -0.11 -2.5–1.9 0.90 wc (cm) 95 (85–99.25) 89 (75–94) -5 -11–2 0.18 years of experience (years) 12.5 (4.25–24.75) 35 (25–39) 18 12–24 < 0.001 duration of menopause (years) 9 (5–15.5) 8 (6–10) 0 -5–3 0.73 systolic pressure (mmhg) 128 (118–137.25) 121 (115–130) -5 -12–2 0.20 diastolic pressure (mmhg) 79.5 (74.88–85.25) 78 (72–84) -2 -6.5–2.5 0.41 years of arterial hypertension 8 (2.8–11) 9 (6–13.5) 2.5 0.54 ci – confidence interval, *hodges–lehmann median difference, †mann–whitney u test seemedj 2021, vol 5, no. 2 serum ferritin levels, arterial hypertension and shift work in women 32 southeastern european medical journal, 2021; 5(2) table 4. age, bmi, years of experience and blood pressure levels in relation to groups, according to ferritin levels (quartile) median (interquartile range) p* < 26.11 (n = 17) 26.12–47.74 (n = 17) 47.75–93.5 (n = 17) > 93.5 (n = 16) q1 q2 q3 q4 age (years) 45 (37.5–53) 57 (49–61) 58 (48–61.5) 58.5 (53.5–61.8) 0.006† bmi (kg/m2) 26.4 (22.65–28.44) 27.3 (24.34–29.9) 26.8 (24.5–31.2) 27.3 (26–30.3) 0.70 wc (cm) 87 (72–94) 88 (77.5–96) 94 (85–102.5) 92 (80.8–97) 0.50 years of experience (years) 21 (7–26) 26 (8.5–37) 35 (17.5–38) 31.5 (14.3–40) 0.08 duration of menopause (years) 4.5 (2.5–8) 7 (4.5–12) 9 (7.3–15.8) 8.5 (7–10.3) 0.16 systolic pressure (mmhg) 120 (106.5–126.5) 128 (119.5–133) 128 (118–141.8) 119.5 (114.3–131.8) 0.08 diastolic pressure (mmhg) 73 (65–80.5) 81 (73–84.5) 80 (74.8–84.5) 79.8 (73.8–87.5) 0.16 years of arterial hypertension 5 (n =1) 6 (2–11.8) 11 (9–12) 11 (8–15) 0.30 *kruskal–wallis test (conover post-hoc) †at the confidence level p < 0.05 there were significant differences in q1 vs. q2; q1 vs. q3; q1 vs. q4. arterial hypertension was present in 22 respondents, 2 of whom (9.1%) had ferritin concentrations in the lowest quartile (< 26.11 ng/ml). there were 45 respondents without arterial hypertension, of which 15 (33.3%) had serum ferritin concentrations in the lowest quartile (< 26.11 ng/ml). likewise, there was no statistically significant difference between ferritin and the presence of arterial hypertension (figure 1). figure 1. distribution of respondents according to ferritin levels and the presence of arterial hypertension ah – arterial hypertension the correlation coefficient of ferritin with other biochemical values was evaluated using spearman’s correlation coefficient. in the group of all respondents, there was a significant and positive relationship between ferritin and crp levels, i.e. the higher the crp levels, the higher the ferritin levels (rho = 0.401; p = 0.001). among respondents who regularly work 8 hours, there 0 10 20 30 40 50 60 70 80 90 100 q1 (< 26.11) q2 (26.12–47.74) q3 (47.75–93.5) q4 (> 93.5) r e sp o n d e n ts ( % ) no ah ah seemedj 2021, vol 5, no. 2 serum ferritin levels, arterial hypertension and shift work in women 33 southeastern european medical journal, 2021; 5(2) was no significant association of ferritin and other indicators, while in the group of those who work in shifts, there was a significant and positive association of ferritin and crp (rho = 0.468; p = 0.002) (table 5). table 5. association of ferritin and other biochemical values between the groups ferritin* p value all respondents tc (mol/l) 0.195 0.11 hdl (mmol/l) 0.103 0.41 ldl (mmol/l) 0.163 0.19 tg (mmol/l) 0.161 0.19 bg (mmol/l) -0.102 0.42 hba1c (%) 0.072 0.56 crp (mg/l) 0.401 0.001 vitamin d (nmol/l) -0.013 0.92 regular daytime work tc (mol/l) 0.232 0.28 hdl (mmol/l) 0.039 0.86 ldl (mmol/l) 0.281 0.18 tg (mmol/l) -0.063 0.77 bg (mmol/l) -0.119 0.58 hba1c (%) 0.227 0.29 crp (mg/l) 0.291 0.18 vitamin d (nmol/l) 0.084 0.70 shift work tc (mol/l) 0.178 0.26 hdl (mmol/l) 0.139 0.38 ldl (mmol/l) 0.109 0.49 tg (mmol/l) 0.215 0.17 bg (mmol/l) -0.086 0.59 hba1c (%) -0.027 0.86 crp (mg/l) 0.468 0.002 vitamin d (nmol/l) -0.068 0.67 *spearman’s rank correlation coefficient (rho) spearman’s correlation coefficient was used to assess the relationship between the number of years of employment in shift work with blood pressure and ferritin levels, and a positive correlation was found between number of years of employment in shift work and systolic pressure, i.e. higher systolic pressure was observed in those respondents who worked longer in shifts (rho = 0.424, p = 0.03) (table 6). seemedj 2021, vol 5, no. 2 serum ferritin levels, arterial hypertension and shift work in women 34 southeastern european medical journal, 2021; 5(2) table 6. relationship of shift work length with blood pressure and ferritin length of work in shifts* p value systolic 0.424 0.03 diastolic 0.202 0.34 ferritin (ng/ml) -0.005 0.98 *spearman’s rank correlation coefficient (rho) discussion this study was conducted to determine the relationship between serum ferritin levels, arterial hypertension and shift work in women. this study demonstrated the persistent link between shift work and systolic blood pressure. a positive correlation between serum crp and ferritin levels was also demonstrated in all respondents, and especially in shift workers. the reason for the expected higher levels of arterial pressure in shift workers, in addition to changes in the circadian rhythm, is increased exposure to psychophysical stress that leads to an inflammatory response [16, 23]. due to the rapid modernization of many sectors, there is a growing need for 24-hour availability of the healthcare system, which creates the need for shift work. according to a 2010 european union report, 23% of men and 14% of women work in shifts [24]. the consequences can be different, but the most significant ones are those that affect the cardiovascular system. previous research has confirmed that shift workers, compared to regular daytime workers, have a higher incidence of ischemic heart disease and myocardial infarction, and more commonly develop arterial hypertension [25, 26]. of noncardiac consequences, a higher incidence of gastric ulcer, obesity, and diabetes has been observed [27–29]. in previous studies, a proportional association between crp levels and blood pressure was confirmed, i.e. higher levels of the inflammatory marker are associated with higher blood pressure levels [30–31]. in the framingham offspring study, rutter and colleagues showed that elevated crp is associated with a higher risk of insulin resistance and metabolic syndrome in women [32]. such a phenomenon can be explained by the fact that higher crp levels are present in conditions that represent risk factors for development of arterial hypertension, such as obesity, metabolic syndrome, smoking and arterial stiffness [33–36]. crp additionally reflects chronic low-grade inflammation of the arterial wall at the site of atherosclerotic plaque [37]. the mechanism of action is mediated by increased expression of adhesion molecules with receptor function, such as vascular adhesion molecule-1 (vcam-1) and intercellular adhesion molecule-1 (icam-1), on endothelial cells of blood vessels [38]. receptors have chemotactic function for macrophages which, by infiltrating the vessel wall, lead to an inflammatory reaction, mediated by proinflammatory cytokines – interleukin-1, interleukin-6 and tumour necrosis factor. furthermore, crp binds to the cell membrane of damaged cells, activating the inflammatory response, which leads to cell dysfunction and atherosclerosis by reducing the synthesis of nitric oxide [39, 40]. there is growing evidence that oestrogen depletion during menopausal transition promotes a systemic inflammatory condition. this condition is characterized by systemic proinflammatory cytokines derived from reproductive tissue, as well as by changes in the cellular immune profile [41]. an inverse relationship between oestrogen and ferritin concentrations is to be expected, i.e. a decrease in oestrogen concentration due to the weakening of ovarian function leads to an increase in ferritin levels in the absence of menstrual bleeding [42]. according to the results of the research by milman and kirchhoff, it follows that during the perimenopausal period, iron stores increase 2–3 times, i.e. an increase in seemedj 2021, vol 5, no. 2 serum ferritin levels, arterial hypertension and shift work in women 35 southeastern european medical journal, 2021; 5(2) serum ferritin concentration by 1 μg/l corresponds to 120 μg of stored iron per kilogram of body weight [43]. just like crp, ferritin is a good indicator of the inflammatory response in the body, reflecting the degree of acute and chronic inflammation [44]. however, serum ferritin levels are directly linked to the occurrence of insulin resistance and diabetes mellitus, especially if it is accompanied by elevated crp levels [45]. although the obtained results are in line with previous findings, the authors point out four important limitations of the research. first, of the respondents included in the research who were in the group of regular daytime workers, 91.7% had previously worked in shifts with a median duration of such work of 18 years (interquartile range 11.5–26.0). second, cross-sectional testing cannot establish a causal relationship, which is why large population and prospective studies are needed. third, as ferritin is an acute-phase protein, it may be elevated in inflammatory or other chronic events that may have remained unrecognized prior to the inclusion of respondents in the study. finally, the uneven distribution of respondents, i.e. a higher share of postmenopausal women working in shifts, is possibly a consequence of personal financial circumstances. conclusion the obtained data show a significant correlation between the length of work in shifts and systolic blood pressure levels, as well as a connection of shift work with higher ferritin and crp levels. further studies on a large sample are needed to determine the association between serum ferritin levels and arterial hypertension. to the best of the authors’ knowledge, this is the first study conducted on women who work in shifts in the healthcare system. the question for further research remains whether the length of shift work should be limited and whether shift workers should undergo more frequent medical examinations due to the increased risk of adverse cardiovascular events. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare references 1. kibel a, belovari t, drenjancević-perić i. the role of transferrin in atherosclerosis. med hypotheses. 2008;70(4):793-7. 2. powell lw, george dk, mcdonnell sm, kowdley kv. diagnosis of hemochromatosis. ann intern med. 1998;129(11):925-31. 3. li j, wang r, luo d, li s, xiao c. association between serum ferritin levels and risk of the metabolic syndrome in chinese adults: a population study. plos one. 2013; 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5(1) review article underactive bladder syndrome: an often overlooked condition 1 ivan radoja department of urology, university hospital centre osijek, osijek, croatia, faculty of medicine, josip juraj strossmayer university of osijek corresponding author: ivan radoja, ivan.radoja@yahoo.com received: jan 30, 2021; revised version accepted: mar 12 2021; published: apr 28, 2021 keywords: urinary bladder, underactive; urodynamics, urinary retention, ultrasonography abstract urinary bladder outlet obstruction can cause lower urinary tract symptoms in men and women, such as a weak or interrupted urine stream, straining to void, hesitancy, and the feeling of incomplete bladder emptying. these symptoms can also be associated with underactive bladder syndrome. this urological condition has a high prevalence in both genders, especially in older age groups. the most common risk factors that can contribute to the pathogenesis of underactive bladder syndrome are aging, detrusor myopathy, neuroinflammatory, neurodegenerative and other lesions of the central and peripheral nervous system, and diabetes mellitus. reduced detrusor contractility, or detrusor underactivity, is the most common pathology of underactive bladder syndrome. detrusor underactivity is a urodynamic definition provided by the international continence society. the urodynamic trace is characterised by a low urine flow rate accompanied by low detrusor pressure or brief detrusor muscle contraction. underactive bladder syndrome is often recognised and treated poorly in clinical practice because the symptoms are not specific. furthermore, underactive bladder can be asymptomatic or coexist with bladder outlet obstruction and overactive bladder syndrome. the aim of this study was to review recent research concerning the aetiology, classification, diagnostic evaluation, and available treatment methods for patients with symptoms of underactive bladder syndrome. making an accurate underactive bladder diagnosis is challenging because it is a multifactorial condition with various patterns of manifestation. consequently, there is still no general agreement and standardisation regarding the most favourable diagnostic and therapeutical approach. (radoja i. underactive bladder syndrome: an often overlooked condition. seemedj 2021; 5(1); 109121) seemedj 2021, vol 5, no. 1 management of underactive bladder syndrome 110 southeastern european medical journal, 2021; 5(1) introduction the storage and excretion of urine in women and men depend on the coordinated activity of the neural centres, smooth muscles of the bladder and urethra, and the urethral striated muscle (1). various urological and neurological diseases can cause voiding dysfunction, which manifests with lower urinary tract symptoms (luts), clinical signs, and syndromes (2). straining to void, a weak urine stream, hesitancy, and other luts during the voiding phase of micturition are prevailing symptoms that urologists encounter in an outpatient and clinical setting. these luts are more often present in men than in women and are commonly associated with bladder outlet obstruction (boo) (3). benign prostatic hyperplasia (bph) and urethral stricture are the usual suspects in men, while in women, the most common causes of boo are functional sphincteric obstruction, antiincontinence surgery, urethral stricture and pelvic organ prolapse (4, 5). these symptoms may be a consequence of detrusor underactivity (dua) (6). dua is caused by reduced detrusor contractility, which can be idiopathic or result from various neurological, urological, autoimmune, muscular, and other diseases (7, 8). according to the international continence society (ics) terminology, dua is a diagnosis based on urodynamic investigations (9). dua is characterised by a low urine flow rate accompanied by low detrusor pressure, brief detrusor contraction, or absent detrusor contraction (10). the result of this is prolonged micturition, failure to achieve complete bladder emptying in an expected time interval, and a high bladder post-void residual volume (pvr). underactive bladder syndrome (uab) is a contemporary clinical term that describes symptomatic dua as a cause of voiding dysfunction (11, 12). uab is defined as a complex of voiding symptoms, such as a weak urine stream, straining to void, and hesitancy (13, 14). storage and post-micturition symptoms, such as a reduced sensation of bladder filling, terminal dribbling, and a feeling of incomplete bladder emptying may also be present, with or without pvr (15). it can be challenging to distinguish if boo or uab is the problem because these conditions have similar symptoms and they can coexist and intertwine, especially in men with bph (16). uab is not so popular when compared with overactive bladder syndrome (oab) and these two syndromes may be present at the same time (17). detrusor overactivity (doa) is another urodynamic diagnosis and oab represents symptomatic doa (18). doa is characterised by involuntary detrusor contractions during the filling phase, which can be spontaneous or provoked during a urodynamic study (9). oab consists of storage luts and it is defined as urinary urgency, with or without urgency incontinence, usually with increased daytime frequency and nocturia, if there is no proven infection or obvious pathology (19). we are better acquainted with the pathophysiology and diagnostic approach of oab compared to uab (20). also, oab treatment methods are more efficient and safer (21). storage, voiding and postmicturition luts are associated with significant morbidity and mortality of men and women aged 65 and over. for example, nocturia associated with bph and oab can lead to a fallrelated bone fracture, persistently high pvr associated with uab can cause bilateral hydronephrosis, chronic cystitis, overflow incontinence, and formation of bladder stones, which can seriously affect the quality of life (qol) (22-25). despite new research initiatives and the latest information about uab and dua, there is still no consensus on a diagnostic and therapeutical approach. furthermore, assessment can be different in men and women. if we could reach a general agreement on diagnosing uab and dua, then we could establish the parameters of non-invasive assessment tools, for example, a combination of uab symptom score questionnaire, ultrasound measurement of detrusor wall thickness, and uroflowmetry result (26). this could help us distinguish patients with uab and dua from those with boo and bph. the aim of this review was to describe the clinical and urodynamic aspects of uab and to emphasise the latest insights in the diagnostic seemedj 2021, vol 5, no. 1 management of underactive bladder syndrome 111 southeastern european medical journal, 2021; 5(1) evaluation and available uab treatment methods. prevalence of underactive bladder syndrome the prevalence rates vary because uab is often a poorly recognised condition in clinical practice, frequently mistaken for boo, and usually not associated with a possible neurological condition. the reason for this is that there is no classification of uab and a definitive diagnosis can be achieved only by conducting a urodynamic study. according to previous research, uab has a high prevalence in both genders, especially in older age groups. uab has the potential of being a serious public health problem in the future because it is expected that the percentage of people aged 65 and over will approach 20% of the total population by 2050 (13, 27). also, neurodegenerative disorders and other neurologic conditions that can lead to luts and uab increase in prevalence with age (28). the prevalence of uab in men under the age of 50 ranges from 9% to 28% and increases with age, affecting 48% of men over the age of 70 (6). the prevalence of uab is approximately 12% in younger women and 45% in older female patients (29). one epidemiological survey, which included a total of 633 participants aged 60 and over, showed that 23% of them had voiding and post-micturition luts, with no difference regarding gender or age; however, only 11% of the participants heard about uab (30). in another original scientific study, urodynamic testing was performed on 1,179 patients with luts aged over 65. after the evaluation of the results, it was established that out of 40.2% of men and 13.3% of women who had dua, 72.6% of the women with dua had doa or stress urinary incontinence (sui) and 46.5% of men with dua also had doa or boo (31). aetiology of underactive bladder syndrome there are many diseases that alone or combined cause bladder innervation disorders and/or damage to the detrusor muscle. this can lead to the inability of the detrusor muscle to generate sufficient intravesical pressure to empty the urinary bladder. therefore, the aetiology of uab is multifactorial and the most important aetiological factors are age-related factors, neurological disorders, boo, and diabetes mellitus (dm) (32). if there is no specific cause, uab can be classified as idiopathic, which is more common in younger patients (33). older people are more likely to develop uab symptoms. the amount of acetylcholine esterase-positive nerves in the bladder decreases with age and a reduced response to bladder filling in the insular cortex occurs (34, 35). also, the proportion of collagen fibres increases with age and the number of smooth muscle cells and muscarinic receptors decreases (34, 36). neurological diseases account for the largest share of risk factors for lower urinary tract disorders such as oab and uab. patients could have luts but no formally diagnosed neurological condition. luts could be an early symptom in the manifestation of an underlying neurological disease, which is referred to as “occult neurology” (37). if we fail to identify an undiagnosed neurological aetiology, we risk poor outcomes for luts treatment. oab symptoms are more common in patients who have had an acute cerebrovascular accident (cva) and in patients with multiple sclerosis (ms) and parkinson’s disease, but uab symptoms and high pvr have also been reported (38-40). urinary retention and dua can occur in early stages of guillain-barré syndrome, but longterm luts are not so common. traumatic, degenerative and other spinal cord injuries can also cause voiding dysfunction. symptomatology depends on the spinal cord injury level. spinal cord injuries below the sacral micturition centre (smc), which is located in the intermediolateral grey of the sacral cord from s2-s4, are most often partial, resulting in weakened bladder contractility and uab symptoms. partial or complete injuries above the smc and neuroinflammatory and neurodegenerative diseases of the central nervous system often result in the appearance of oab symptoms. infectious diseases, such as seemedj 2021, vol 5, no. 1 management of underactive bladder syndrome 112 southeastern european medical journal, 2021; 5(1) neurosyphilis, herpes zoster, herpes simplex, and acquired immunodeficiency syndrome, are also associated with a neurological dysfunction that causes the symptoms of uab and/or oab (41-44). radical prostatectomy, radical hysterectomy, and other pelvic surgical procedures, in which peripheral nerve damage may occur, are examples of an iatrogenic cause of uab (35). even oab and boo can cause structural changes of the urinary bladder over time, which include a reduction in detrusor blood flow, detrusor muscle deterioration, and proliferation of connective tissue, which can lead to dua and uab (45). dm is one of the most common diseases in general population, which has recently reached epidemic proportions in many countries and is associated with numerous complications and organ damage (46). one of the more common complications of dm is diabetic cystopathy (dc) (47). the prevalence of dc ranges from 20 to 90% (48). as with uab, we still lack a standardised diagnostic assessment of dc. the symptoms include an impaired sensation of bladder fullness, reduced bladder contractility, increased bladder capacity, and increased pvr. dc can be asymptomatic early in the course of dm (49). dc is a result of sensory and autonomic polyneuropathy, microvascular damage, dysfunction of detrusor smooth muscle cells, and dysfunction of urothelial cells (50). urothelial cells were considered to be just a barrier between the urinary tract lumen and underlying tissues of the urinary tract wall, but they have a more active role in bladder physiology. these cells form a functional unit that responds to external events by releasing adenosine triphosphatase, nitric oxide, and prostaglandins (51, 52). these modulator agents regulate the activity of afferent nerves and the detrusor smooth muscle and process the information about the urinary tract chemical and physical status (e.g. luminal pressure, urine composition) (53). with all this said, it just goes to show that we still have a lot to learn about the function of the lower urinary tract and surrounding tissues. diagnostic evaluation of underactive bladder syndrome the symptoms of uab are not specific and they can overlap with other voiding dysfunctions, especially with boo. the symptoms that may be suggestive of uab include hesitancy or a delay in passing urine, weak or thin urine stream, interrupted urine flow, post-void dribbling of urine, feeling of having to go again after the stream has stopped, voiding a second or even multiple times after the initial voiding has completed, having to strain to facilitate voiding, and frequent urination day and night in small volumes (54). with history-taking and a physical examination in medical practice, we can only assume that an observed patient has uab. it is especially important to consider neurological symptoms and diseases. during a physical examination, we palpate the suprapubic region to check the pain and fullness of the bladder. also, we assess the tone of the anal sphincter, perianal sensation, bulbocavernosus reflex, and plantar reflex, which can be compromised, absent, or significantly diminished (55, 56). some studies have examined the usefulness of noninvasive methods, such as flow pattern on uroflowmetry, intravesical prostatic protrusion (ipp), pvr volume, and bladder voiding efficiency (bve) to distinguish between uab and boo (15, 57). non-invasive methods are very useful for the assessment of patients who cannot go through invasive urodynamic testing. uroflowmetry is a simple and non‐invasive urodynamic test of measuring and recording the voided volume, urinary flow rate, and wave shape throughout micturition (58). it is a screening procedure and its results may be indicative of boo or uab. ipp is measured during almost every examination of men with luts. it is measured using the transabdominal ultrasound in the sagittal view and defined as the vertical distance from the tip of the protrusion to the base of the prostate (59, 60). regarding this distance, ipp can be quantified into three grades, which more or less correlate with the symptoms related to boo: grade 1 (≤ 5 mm), grade 2 (5-10 mm), grade 3 (≥ 10 mm). pvr is measured immediately after seemedj 2021, vol 5, no. 1 management of underactive bladder syndrome 113 southeastern european medical journal, 2021; 5(1) voiding by ultrasound or by inserting a catheter into the bladder through the urethra. using ultrasound, we measure three dimensions of the bladder: maximal transverse (width), anteriorposterior (height), and longitudinal (length). then we determine the pvr volume using the dicuio’s formula (volume [ml] = height [cm] × depth [cm] × transverse diameter [cm] × 0.52) (61). to determine bve, we measure the bladder volume before voiding (bvvoid) using ultrasound and then we measure voided volume (vv) using a measuring cylinder. using the equation ([vv/bvvoid ] × 100), we can express bve by fraction (%) of the voided volume. a combination of lower ipp (< 8.2 mm) and lower bve (< 70), sawtooth and interrupted wave shape on uroflowmetry might be useful factors to predict du and uab. despite all non-invasive methods, pressure-flow study is practically the only method by which we can establish an accurate diagnosis of uab (62). the parameters observed during this study are peak flow (qmax) and detrusor pressure at peak flow (pdetqmax) (63). using these parameters, we can assess the contractility of the bladder by using the bladder contractility index (bci) formula according to the equation pdet@qmax+5qmax (64). with this formula, we can divide bladder contractility into strong (bci > 150), normal (bci 100–150), and weak (bci < 100). also, we can exclude or confirm the presence of the obstruction in men using the bladder outlet obstruction index (booi) formula according to the equation pdet@qmax-2qmax (65). based on the results of the booi formula, men can be divided into obstructed (booi > 40), equivocal (booi 20−40), and unobstructed (booi < 20). booi formula does not apply to women. at the moment, the blaivas and groutz nomogram is the most common method used to define boo in women as a qmax < 12 ml/sec combined with a pdet@qmax of > 20 cm h2o in a pressure-flow study (66). there is a potential future test for uab that includes urine biomarkers, such as nerve growth factor, and bladder wall biopsy with an electron-microscopic histopathologic ultrastructural assessment of the detrusor (67, 68). the research of these diagnostic methods is still ongoing, so further studies are needed to implement them into clinical practice. treatment of underactive bladder syndrome the treatment of uab is focused on improving the qol and preventing complications. if the patients are asymptomatic, we still need to administer the treatment. also, we must inform patients about possible side effects of each type of treatment. it is necessary to achieve the status of an empty bladder regardless of the aetiology of uab to avoid chronic urinary retention, bilateral hydronephrosis, recurrent urinary tract infections (uti), bladder stone formation, and overflow incontinence. common methods of treatment are timed voiding, double voiding, straining to void, credé or valsalva manoeuvre, clean intermittent catheterisation (cic), pharmacotherapy, and surgical treatment (35, 69, 70). in patients with vesicoureteral reflux and high intravesical pressure, the credé and valsalva manoeuvres are contraindicated. constipation should be avoided by dietary fibre intake and exercise, which are usually recommended as first-line treatment (71). prolonged use of laxatives may lead to some adverse effects. cic must be recommended to patients who have chronic urinary retention with high pvr. if possible, placement of an indwelling urinary catheter and suprapubic cystostomy should be avoided due to complications, such as catheter encrustation, urethral erosion, and increased risk of uti and bladder cancer (72). we need to explain the benefits of cic to each patient to enhance compliance. silicone catheters are most commonly used because they carry a lower risk of allergic reactions. patients can perform catheterisation on their own or they are catheterised by caregivers. recommended daily number of catheterisations ranges between 4 and 6 and the amount of urine discharged at every catheterisation should not exceed 400– 500 ml. silicone catheters are most commonly used. cic is impractical and inconvenient for elderly, visually impaired and mentally handicapped patients, and for patients with seemedj 2021, vol 5, no. 1 management of underactive bladder syndrome 114 southeastern european medical journal, 2021; 5(1) limited manual dexterity. complications of cic are urethral injuries (e.g. false passage, strictures), uti, and haematuria (73). there are no effective oral drugs for uab treatment. contemporary pharmacotherapy of uab includes the use of α-adrenergic blockers to reduce outlet resistance at the level of the bladder neck and muscarinic agonists or choline esterase inhibitors to increase detrusor contraction (74). these drugs can be used as monotherapy or in combination. α-adrenergic blockers, such as tamsulosin at a dose of 0.4 mg once a day and silodosin at a dose of 8 mg once a day, are in most cases considered for the initial stage of uab treatment in men (75). αadrenergic blocker therapy for women with uab is an off-label regimen in most countries (76). bethanechol chloride (bc) and pyridostigmine bromide (pb) are the most common orally administered parasympathomimetic agents used in clinical practice (77). the usual adult oral dose for bc ranges from 10 to 50 mg three or four times a day and from 60 to 180 mg for pb. their effectiveness is limited due to the downregulation of muscarinic receptors and potentially life-threatening side-effects, such as bradycardia and ventricular tachycardia (32). the european association of urology (eau) states that they should not be prescribed for uab treatment (75). prostaglandin e2 (pge2) can increase the inotropic activity in smooth muscles (72). it prevents the release of noradrenaline from sympathetic nerve endings. intravesical administration of pge2 can increase detrusor contractions and decrease maximal urethral closure pressure (75). the effectiveness of pge2 is limited and it is not recommended for routine treatment. onabotulinumtoxin a injections into the external urinary sphincter reduce urethral resistance, eliminate the inhibitory effects of urethral afferent nerves on the detrusor and allow easier voiding with the aid of increased abdominal pressure (72). this therapy has not been approved yet and there is no standard dosage. the efficacy of transurethral resection of the prostate and transurethral incision of the bladder neck in patients with boo and accompanying uab is questionable. patients should be informed that they may not benefit from this type of procedure. chronic urinary retention associated with uab often leads to myogenic decompensation because of an overdistended bladder. reduction cystoplasty decreases the capacity of the bladder and facilitates bladder emptying, but it does not increase bladder contractility (72, 75). this treatment method should be performed only in well-chosen cases and in patients with residual detrusor contractility. sacral neuromodulation therapy (snm) is appropriate for well-chosen patients who have not been helped by more conservative treatments, such as drug therapy (78). snm uses a surgically implanted device to send electrical impulses to the sacral nerves located in the lower back, which modify the function of the detrusor muscle and pelvic floor muscles. it is one of the more expensive methods of treatment and it is performed in specialised high-volume tertiary referral centres. transurethral intravesical electrical stimulation and use of the stem cells are novel treatment methods with reports that they can improve detrusor contractility and increase weak individual myocyte contractility (79). neurovascular latissimus dorsi (ld) detrusor myoplasty is suitable for motivated young patients who do not want to undergo cic. the ld muscle flap is wrapped around the bladder, attached to the pelvic fascia and ligaments, and it is anastomosed to the deep inferior epigastric vessels and lowest motor branches of the intercostal nerve (80). ld detrusor myoplasty can adequately restore bladder emptying on demand (81). further studies are needed to implement these surgical and novel treatment methods in clinical practice. regardless of the type of treatment used, patients should have frequent follow-ups with repeated urodynamic examination where necessary. discussion ics conducted a comprehensive review of the terminology pertaining to the lower urinary tract function and dysfunction and recommended the use of the term dua (9). uab has not yet been seemedj 2021, vol 5, no. 1 management of underactive bladder syndrome 115 southeastern european medical journal, 2021; 5(1) defined by the ics. furthermore, the current definitions of both conditions do not include possible aetiological risk factors. based on the aetiology, uab can be classified into four types: neurogenic, myogenic, and idiopathic (29). neurogenic uab is associated with changes in the afferent and efferent signals of the micturition reflex (82). myogenic uab is associated with modified contraction mechanisms of detrusor muscle cells, which can result in reduced autonomous activity of the bladder (83). uab can be the combination of myogenic and neurogenic factors or a combination of two or more neurogenic factors. idiopathic uab has an unknown cause or mechanism of apparent spontaneous origin (84). based on urodynamic findings, uab can be classified into three types: du, acontractile detrusor, and oab/uab combination (29). patients with dua have impaired detrusor contractility and can void incompletely or they have no urine flow during the urodynamic examination. if there are no detrusor contractions during cystometry and a pressureflow study, then we are talking about an acontractile detrusor. it is an extreme type of dua, patients have high pvr, and the emptying of the bladder is performed by increased abdominal pressure or cic. it is important to note that some patients have uab and oab symptoms (17). if we suspect this is the case, a urodynamic assessment is mandatory. doa is recorded during filling cystometry and dua during a pressure-flow study. education about the symptoms and clinical signs of the lower urinary system is of utmost importance for all urologists, urogynecologists, and neurourologists. taking a medical history in an outpatient setting can be complicated at times and for this reason, it is recommended to use questionnaires, frequency-volume charts, and bladder diaries in the evaluation of symptoms. physicians must also be well acquainted with performing a physical examination, especially of neurological status. urodynamic testing is very demanding and one must be very familiar with the methodology to adequately interpret the results. catheter placement and recording of urodynamic traces must be performed according to the ics standards. there is still no effective pharmacotherapy of uab. for now, cic is the only effective therapy for achieving empty bladder status, but unfortunately, not all patients are willing to perform the catheterisation procedure multiple times a day for an extended period of time. all patients should be offered treatment taking into account the cause of uab and the most distinct symptomatology. cic is the main treatment method in the management of patients with high pvr and uab. contemporary research has shown that there are drugs which can have a positive effect on dua, but in most cases, these studies were not randomised clinical trials. oral parasympathomimetics can improve detrusor contractions and facilitate bladder emptying, but they do not have a long-lasting effect and they have many serious side effects. ics and eau guidelines do not recommend or approve the use of parasympathomimetics for uab. this therapy is contraindicated in patients with coexisting oab and uab symptoms because it may worsen bladder emptying, leading to urine retention and uti. therefore, we must perform a thorough neurourologic and urodynamic assessment if we suspect that there are multiple voiding dysfunctions in one patient at the same time. α-adrenergic blockers are useful in men with boo accompanied by pvr, but their use in women with uab is not supported by the guidelines. snm and reconstructive surgical methods are expensive and usually performed in experienced high-volume centres. a large number of patients diagnosed with uab sometimes do not have access to such centres due to distance or cost of treatment. research is underway on possible new pharmacotherapeutics and new surgical methods of treating uab. conclusion regardless of the complexity of the neuromuscular mechanism of controlling the storage of urine and emptying the bladder, we take the process of voiding for granted. when some form of voiding dysfunction occurs, patients experience how much it can affect the seemedj 2021, vol 5, no. 1 management of underactive bladder syndrome 116 southeastern european medical journal, 2021; 5(1) qol (85). uab is a relatively new term of voiding dysfunction and was recognised in some aspects years before we knew what it is today. it has a high prevalence in the elderly population (86). despite new insights, uab is still shrouded in mystery (6). many diseases can cause the loss of sensation in the bladder and/or detrusor muscle damage (8). it is a great challenge to determine exactly which disease caused the symptoms of uab. we must do our best to identify the most likely aetiology of uab in each patient. classification of uab needs to be improved to facilitate diagnostic processing and it should be based on aetiology, urodynamic findings, and symptoms. at this point, we have a great knowledge about the symptoms of uab and about the urodynamic characteristics of dua. this can improve through further research on the pathophysiology of uab. future developments in the pharmaceutical industry, gene therapy, and biomedical applications are expected to close the gap in treatment. we need to raise awareness among those patients who are highly likely to develop uab in order to initiate treatment at the right time and avoid complications. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. yu yd, jeong sj. epidemiology of underactive bladder: common but underresearched. investig clin urol. 2017; 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1(2) progins mutation of progesterone receptors and its role in premature birth – an overview 1 mirta kadivnik1, andrijana muller1,2, iva milić vranješ1, siniša šijanović1,2, jasenka wagner3 1 department of gynecology and obstetrics, osijek university hospital, osijek, croatia 2 department of gynecology and obstetrics, faculty of medicine, josip juraj strossmayer university of osijek, croatia 3 department of biology and medical genetics, faculty of medicine, josip juraj strossmayer university of osijek, croatia, department of chemisttry and biochemistry, faculty of dental medicine and health, josip juraj strossmayer university of osijek, croatia corresponding author: mirta kadivnik, md – mirta.kadivnik@gmail.com received: september 21, 2017; revised version accepted: november 24, 2017; published: november 24. 2017 keywords: drinking water, isotopes, radioisotopes abstract premature birth (prior to 37 weeks of gestation) is a big medical and socioeconomic problem. it accounts for 8-12% of the total number of births, and apart from causing increased mortality of newborns, it is also the cause of increased morbidity. fifteen million babies per year are born preterm. despite the frequency, consequences and costs of premature delivery, very little has been done for preventing it, especially for preventing extremely premature deliveries (before the 28th gestation week). etiology of premature labor is multifactorial, and includes pathophysiology, genetic and environmental factors. recent scientific research shows that genetic factors, mostly present in the mother's genome, account for up to 40% of variation in the delivery time. it is believed that premature birth exhibits the same cascade of events like a normal birth, only it starts sooner. this process is controlled by a series of hormonal effects between the fetus, the placenta and the mother. one of the key signaling pathways in this series is the progesterone signaling pathway. progins allele is a progesterone receptor gene modification. it is made of three variants: v660l, h770h and alu insertion. progesterone receptors with progins mutation are less susceptible to progesterone activity, and it seems that the withdrawal of progesterone causes the beginning of birth cascade. mutation of +331 g/a progesterone receptor is a newly discovered mutation. it is believed that this mutation leads to a pr-a and pr-b receptor quantity disorder before the delivery term. the aim of this review is to summarize all recent knowledge about progins and +331 g/a mutation of progesterone receptors and to estimate whether this genetic mutation has a value in modulation of risk of preterm birth. seemedj 2017, vol 1, no. 2 progins mutation of progesterone receptors and its role in premature birth… 56 southeastern european medical journal, 2017; 1(2) introduction premature or preterm birth is recognized as a worldwide problem. the percentage of preterm births has not been reduced despite existing research and therapy. it is defined as birth between 22nd and 37th week of gestation, and it is one of the major causes of prenatal mortality and morbidity. prematurity accounts for 70% of neonatal mortality and 75% of neonatal morbidity (1). according to gestational age, preterm birth is divided to extremely preterm birth (between 24th and 28th week of gestation), early preterm (between 28th and 34th week of gestation) and late preterm birth (between 34th and 37th week of gestation) (2). the percentage of preterm births varies from 5% of all deliveries in europe to 18% in south african area (3). there are also variations of the percentage of preterm birth in diverse ethnical groups; for example, the percentage of preterm births is bigger in the population of african americans than in the population of caucasians (4,5). in 2010, there were around 15 million preterm births in the world (3,5). in the same period in croatia, the percentage of preterm births was between 5.19 % and 7.88 % of all births, with a tendency of increasing since 2008 (6). etiology and risk factors of preterm delivery etiology of preterm birth is heterogenic and is connected to different metabolic pathways in the human body. for almost 50 % of preterm births, the cause is unknown. preterm birth is divided in three subtypes (2): • spontaneous preterm birth (spontaneous start of labor); 50% of all preterm births, occurring more frequently in the population without any risk factor for preterm birth • preterm premature rupture of membranes (pprom); 25% of all preterm births, occurring more often in the african-american population, in most cases as a result of infection • iatrogenic preterm birth before 37 weeks of gestation, due to maternal or fetal medical reasons, or other nonmedical reasons that could jeopardize the health of mother and/or fetus (e.g., preeclampsia, placenta previa, placental abruption, multiple gestation, grow restriction of fetus); 25% of all preterm births. there are many risk factors that relate to preterm birth, as indicated in table 1. in an ideal situation, risk factors for preterm birth should be identified prior to or during the first trimester of pregnancy and, if possible, that should lead to interventions which would result in term delivery. some pathways of starting a preterm delivery and related risk factors are presented in table 2. nowadays it is more common to use the term “preterm parturition syndrome” (7), or even “great obstetric syndrome”, because of its multifactorial etiology. there are four major factors leading to preterm labor (7,8,9,86): 1) pathological uterine distension; 2) maternal fetal stress (premature activation of the maternal or fetal hypothalamicpituitary adrenal axis); 3) abruption (decidual hemorrhage) and 4) infection / exaggerated inflammatory response. all these processes could lead to cervical shortening and could start long before obvious signs of preterm birth are shown (10). also, although they start at different ends of pathophysiology of preterm birth, they end in the same way, by activating choriodecidual reaction, uterine contractility and changes of the cervix. all these changes lead to preterm birth. (kadivnik m, muller a, milić vranješ i, šijanović s, wagner j. progins mutation of progesterone receptors and its role in premature birth – an overview. seemedj 2017; 1(2); 55-70) seemedj 2017, vol 1, no. 2 progins mutation of progesterone receptors and its role in premature birth… 57 southeastern european medical journal, 2017; 1(2) pathological uterine distension it is highly possible that uterine overdistension (caused by multifetal pregnancy, polyhydramnios or any other cause of uterine distension) can cause expression of contractionassociated proteins (caps) in the myometrium. overdistension of the uterus also induces formation of gap junctions, upregulates oxytocin receptors and produces prostaglandins and inflammation cytokines (11). all this can initiate events that could change timing of uterine activation and lead to uterine contractions and cervical dilatation. maternal-fetal stress (premature activation of the maternal or fetal hypothalamic-pituitary adrenal axis) in situations of stress, some circumstances disturb normal functions of a person. maternal stress (infection, multiple pregnancies or psychological stress, such as depression or anxiety) can activate maternal hpa axis and cause preterm birth. premature fetal hpa activation, on the other hand, can be the result of stress of uteroplacental vasculopathy. it is more highly correlated with preterm birth than mother’s stress (12). the main pathway of this cause seems to be a change in fetal adrenal-placental endocrine cascade, which leads to early rise of maternal crh (corticotropin-releasing hormone) and estrogen levels. crh plays a role both in term and preterm birth. usually it is released by the hypothalamus, but during pregnancy it is released by trophoblast and decidual cells, too (13,14). increased production of placental crh stimulates production of acth, which further table 1. etiological risk factors associated with clinical presentation of preterm birth (2). medically induced preterm birth pprom (preterm premature rupture of membranes) spontaneous preterm birth maternal factors gestational hypertension and vascular disorder acute illness or chronic condition obstetrical complication antepartum bleeding maternal age > 35 years fetal factors intrauterine growth restriction unstable fetal condition fetal anomaly multiple pregnancies infection uterine distension cervical anomalies african-american ethnicity disadvantaged population previous preterm birth low body mass, poor weight gain strenuous physical workload, ergonomic factors uterine anomalies psychosocial stress lifestyle, smoking drug abuse maternal age < 18 years unknown table 2. causes and pathological pathways of preterm birth (7) • uterine distension • ischemia • infection • cervical disease • abnormal allograft reaction • allergic phenomena • endocrine disorders seemedj 2017, vol 1, no. 2 progins mutation of progesterone receptors and its role in premature birth… 58 southeastern european medical journal, 2017; 1(2) stimulates production of cortisol. cortisol inhibits hypothalamic crh and acth, but on the other hand, stimulates crh from the placenta. crh also induces production of prostaglandins in the placenta (15). the increase of prostaglandins results in parturition through elevation of proteases in the genital tract (e.g., mmp) and higher myometrial contractility (16). also, prostaglandins influence the pr-a: pr-b ratio and induce functional progesterone withdrawal (17). stress, maternal and/or fetal, can also stimulate steroid-induced immunophilin cochaperone fkbp51 in the decidua. it can also cause functional progesterone withdrawal through inhibition of progesterone receptors (18). one more pathway of preterm birth caused by activation of fetal hpa axis is the estrogen pathway. fetal acth induces synthesis of dhea. dhea in fetal liver is converted to 16-hydroxydhea-s. the placenta converts these precursors to e, e2 and e3, which further activate the myometrium through increase of gap junction formations, oxytocin receptors, activity of prostaglandins and increasing of enzymes responsible for myometrium contractions (e.g., calmodulin) (19,20). all the pathways mentioned above cause contractions of the myometrium and the start of labor. infection / exaggerated inflammatory response it is the only evidence-based and proven cause of preterm birth, which activates different pathways leading to preterm birth. infection activates a cascade in the immunological response of the mother and leads to preterm birth. inflammation is a coordinated process, and its role is basically to protect the host. in a normal situation, when the immune system is properly controlled, inflammation is protective. in other cases, it is harmful. preterm birth can be caused by both systemic and local genitourinary pathogens. in most cases, the cause can be symptomatic or asymptomatic bacteriuria (21), presence of genital infections (22), periodontal disease (23) and clinical and subclinical chorioamnionitis. the last-mentioned infection is the cause of as much as 50 percent of preterm births before 30 weeks of gestation (24). studies have proven that the actual cause of preterm birth is not the infection itself but rather a disorder of maternal immunity (21-24). pathways of preterm birth in infection start by binding of bacterial ligands to toll-like receptors (tlrs) in placental, decidual, amniochorion and cervical cells. tlrs and local leucocytes activate nfkappab, which in turn, starts the maternal and/or fetal inflammatory response. whether tlrs will start the activation of nfkappab or not, depends on the presence of some intracellular signaling adaptors (e.g., myd88), coreceptor molecules (e.g., cd 14) and receptor modulators (soluble il 6 receptor, soluble tnf receptor -1, etc.) (25-27). activation of nfkappab leads to activation of neutrophils, macrophages and various proinflammatory mediators. the most important mediators of this response are tnf and il 1 beta. they induce cox-2 expression and production of prostaglandins. tnf also initiates expression of various mmps in the amnion, chorion, decidua and cervix, and degrade the matrix of cervix and fetal membranes (28,29). tnf alfa can also induce apoptosis in amniotic epithelial cells, which leads to pprom. not just the immune response, but some bacteria themselves (e.g., pseudomonas, staphylococcus, streptococcus) can have a direct role in the pathogenesis of preterm birth. they can produce enzymes that can degrade fetal membranes, as well as phospholipase a2 and endotoxin, which stimulate uterine contractions (30). abruption / decidual hemorrhage vaginal bleeding caused by decidual hemorrhage is a risk factor for preterm birth and pprom. according to one study, vaginal bleeding lasting more than one trimester increases the risk of pprom seven times (31). pprom develops after decidual hemorrhage, due to high concentration of decidual tissue factor. it combines with factor viia of hemostasis, seemedj 2017, vol 1, no. 2 progins mutation of progesterone receptors and its role in premature birth… 59 southeastern european medical journal, 2017; 1(2) and in the end, thrombin is generated. thrombin binds to decidual protease-activated receptors, which induce expression of proteases (e.g., mmp). abruption can also be related to an inflammatory reaction without infection. it starts as a result of activation of the immune response by free hemoglobin chains and protease. pathological cervical change most cases of cervical changes prior to term pertain to cervical insufficiency. the changes that cause preterm birth may be the result of a congenital disorder, post-surgical trauma or damage caused by trauma. in most cases, though, cervical shortening is the result of inflammatory or hemorrhagic pathways. as mentioned above, main changes in preterm birth happen on the placental level. they include effects on the level of prostaglandins (immunology and infection pathways) and the endocrine level (progesterone/estrogen pathway). recently, new pathways have been identified and they seem to be connected with biological and psychosocial factors. these pathways include, as mentioned above, influence of genetic factors, factors of stress, factors that can be attributed to the mother or to the fetus, conditions which cause mechanical stimuli, and cases of inflammation and infection. the role of genes in preterm birth investigations about genetic influence on preterm birth have been common during the last two decades. studies have shown that the risk of preterm birth is higher in women born prematurely. women who have had a previous preterm birth are at greater risk to have it again. after the first preterm delivery, the chance of another preterm delivery in the same mother goes up to 30-50% (32). also, it has been shown that mothers whose sisters, mothers or female cousins have had a preterm birth bear greater risk of having a similar preterm birth themselves (33,34,35). an extensive study, conducted in sweden in 2010, showed that both maternal and fetal genes are involved in preterm birth (36,37,38). fetal genetic factors accounted for 13.1% of the variation in gestational age at delivery, while maternal genetic factors accounted for 20.6% (37). in a similar study, estimation of the percent of variation connected to fetal genetic factors ranges from 11% to 35%, while the range for the maternal genetic contribution is 13-20% (39). another study, by svensson et al., showed that 25% of variation in preterm birth was explained by maternal genetic effects, 5% by fetal genetic effect, 18% by the environment created by the couple and 52% by unshared environmental effect (40,41). this and other studies showed that paternal genetic influence on preterm birth is minimal (up to 5%) or there is no influence at all. that fact is in discrepancy with two norwegian studies (42,43). in the same study by svensson et al. it is shown that fetal genetic effect is higher if preterm birth is induced for medical reasons. genes can influence different pathways in the body connected with preterm birth. most of the studies involved gene contribution in immunology and inflammation pathways (32,44). induction of proinflammatory mediators, especially tnf and its receptors, has been suggested to have a crucial role in activation of labor, both term and preterm (45). proinflammatory and anti-inflammatory cytokines (interleukins) have also been investigated (46,47). there have also been investigations on genetic mutations leading to change of uterine contractility and change of cervical tissue, which leads to cervical shortening and spontaneous preterm birth. they involve genetic mutations in dopamine receptors, ost receptors, progesterone pathway, etc. recently there have been studies about stress influence and genetic mutations in preterm birth (48). a big genome-wide association study of a large cohort of women of european ancestry has shown that maternal variants at the ebf1, eefsec, agtr2, wnt4, adcy5, and rap2c loci were associated with gestational duration, and that maternal variants at the ebf1, eefsec, and agtr2 loci were associated with preterm birth (49). in this review, the authors will try to see the influence of specific genetic polymorphisms connected to progesterone pathway that has an seemedj 2017, vol 1, no. 2 progins mutation of progesterone receptors and its role in premature birth… 60 southeastern european medical journal, 2017; 1(2) important role in maintenance of pregnancy and changing contractility of the uterus. the role of progesterone in onset of preterm birth progesterone (p4) is one of essential hormones in establishing and maintaining pregnancy. it is a 21-carbon steroid hormone which is mainly produced in the ovaries, placenta, brain and the adrenal glands (50). in early pregnancy, it is produced by corpus luteum whereas from 7th week of pregnancy onwards, its production occurs in the placenta. progesterone is required for maintenance of pregnancy and one can see its influence on uterus contractility by inhibition of cervical ripening and decreasing of the production of chemokines. progesterone also prevents apoptosis in fetal membranes in both basal and proinflammatory conditions, and it prevents pprom and, consequently, preterm birth (51,52). progesterone produces its physiological effects through progesterone receptors (pgrs). pgrs are expressed in the central nervous system, ovaries, breasts, and the female reproductive tracts, including the vagina, cervix, fallopian tubes and uterine endometrium and myometrium. at term, depending on the species, either withdrawal of p4 by a decrease in hormone levels or alteration of pgr signaling relieves the suppression of inflammation and contraction, which allows the myometrium to start contractions and lead toward labor. multiple mechanisms, including p4 metabolism, regulation of pgr gene expression, pgr posttranslation modifications and pgr co-regulators, which mediate or regulate uterine p4/pgr signaling, have been identified and reviewed (53). one of the hypotheses about preterm birth is that the cascade of events in preterm birth is similar as the events at term birth, with the difference being only that it starts earlier. progesterone receptors are members of the group of steroid hormone receptors. they are made of central dna-binding domain (dbd), nterminal part with proximal activation function (af1), distal af3 in b upstream segment, and nuclear localization signal which is found upstream of lbd. af1 is ligand-independent, while af is not. af1 has an influence on direction of transcription (54). there are two types of progesterone receptors: nuclear and membrane receptors (8). the nuclear prs function as ligand-activated transcription factors and they influence gene expression. membrane prs are on the cell surface; they are related to g-protein coupled receptors and single transmembrane receptors, and they appear to mediate direct non-genomic actions of progesterone (8). maintenance of pregnancy is mostly regulated through nuclear progesterone receptors, while membrane progesterone receptors are less sensitive to the influence of progesterone. by influencing prs, progesterone activates a variety of pathways and induces expression of other genes, which leads to activation or deactivation of myometrium. those pathways include activation of certain caps (contractionassociated genes) such as connexin, ion channels (e.g., calcium channels), uterotonin receptor and enzymes that influence synthesis of local prostaglandins (1,55). nuclear receptors are coded by the prostaglandin gene located on chromosome 11 (11q22-q23) (56). two isoforms of nuclear progesterone receptors are most significant for progesterone influence; more specifically, pr-a and pr-b. both receptors have the same dnabinding domain, ligand-binding domain and hinge region. the only difference between these two is the fact that there are additional 165 amino acids present in the n-terminus of pr-b (18). there are some other forms of nuclear prs known, such as prc, prm, prs, prt etc., but their significance in human birth is irrelevant (55). pr–b have the function of transcriptional activators of genes involved in maintaining pregnancy, and pr–a repress the activity of prb. according to literature, most of the prs in the myometrium during pregnancy are pr-b, and as the term of delivery comes nearer, the number of pr-a increases (36). pr isoforms influence the expression of diverse downstream genes through a complex regulatory network which includes the nf-κb, zeb-micrornas and upr pathways, as well as direct transcriptional regulation. they have a seemedj 2017, vol 1, no. 2 progins mutation of progesterone receptors and its role in premature birth… 61 southeastern european medical journal, 2017; 1(2) combined influence on activities of downstream effectors. the overall p4/pgr signalingdependent molecular profiles are modified by activities of pgr isoforms, co-regulators and the ligand availability. p4/pgr signaling mediates and utilizes these interconnected pathways to determine the state of the myometrium throughout pregnancy (53). different influences of pr-a and pr-b isoforms are reflected in their influence on expression of proinflammatory and anti-inflammatory genes. pr-a act proinflammatory, by increasing expression of proinflammatory genes for ptgs2, il8, il1a and ptx3. on the other hand, pr-b inhibit expression of proinflammatory genes (57). in mammals, it has been proven that birth starts at the moment of progesterone withdrawal. however, in humans, measurements of the levels of serum progesterone in the blood at the time of birth showed that there was no declination of progesterone level. at least five pathways of how withdrawals of progesterone influence the start of birth were examined. they include: 1) reduced bioavailability of progesterone, 2) increased cortisol concentration in late pregnancy, which leads to progesterone and cortisol competing for binding to glucocorticoid receptors (58), 3) conversion of progesterone to an inactive form, 4) changes in isoforms of pg receptors (59), 5) changes in progesterone co-regulators (54). some studies also include functional estrogen activation (60) and inflammation resulting in nfkappa mediated pr repression (61). in this short review, the authors have reviewed literature data about changes in isoforms of pg receptors. there is a hypothesis that human parturition involves changes in expression of myometrial nprs and that change of expression leads to functional progesterone withdrawal and start of birth. the main theory is so-called ist (isoform switch theory) (36,62), according to which, as mentioned above, the ratio of pr-a and pr-b is changed in favor of pr-a. pr-a repress pr-b and reduce transcription of pregnancy promoting genes. increasing level of pr-a at the end of pregnancy occurs because of a change in methylation of pr-a promoter region. that eventually leads to pre-term contractility of the uterus. chai et al. wanted to clarify epigenetic mechanisms that contribute to the control of pr isoform expressions in the pregnant human myometrium. they researched the change in methylation of the cpg island in promoter region of prs and uncovered an epigenetic mechanism for elevated pr-a:pr-b expression ratio in term myometrium during progesterone withdrawal. pr-a promoter loses h3k4me3 selective demethylase jaridia, which leads to increased methylation of the pr-a promoter, change of its transcriptional activity and change of pr-a:pr-b ratio (63). in further investigations, nadeem et al. identified a mechanism by which p4 action of maintaining the pregnancy has been withdrawn even in the presence of elevated levels of this hormone in circulation. unliganded pr-a localize to the nucleus, where they paradoxically activate transcription of cx43 gene through interaction with ap 1 heterodimers (64). namely, during pregnancy, under the influence of p4, pr-b create a complex with transcriptional repressors and inhibit transcription of cx43. in labor, as a result of change of pr-a:pr-b ratio, pr-a become unliganded and encourage expression of cx43. a similar mechanism can be applicable on other labor-associated genes, such as ptgs2, oxtr, oxn, ptgds and nfkb2 and some proinflammatory cytokines and matrix proteins. this study has also showed increased level of progesterone metabolized with 20 alpha hsd enzyme, which might be important for the usage of appropriate progesterone for therapy of preterm birth (59). beubaker et al. showed that the repressive activity of pr-a and their amount in the myometrium are increased by pro-inflammatory stimulation (65). seemedj 2017, vol 1, no. 2 progins mutation of progesterone receptors and its role in premature birth… 62 southeastern european medical journal, 2017; 1(2) genetic variants of prs and their influence on preterm birth common variant in human prs is the so-called progins allele. it is present in some frequency in more than 20% of population. progins mutation of progesterone receptors is extensively investigated. one of the research groups, romano et al. (66), showed that it is characterized by a 320 bp pv/hs-1 alu insertion in intron g and two-point mutations, v660l in exon 4 (rs1042838 snp) and h770h (silent substitution) in exon 5 (rs1042839 snp). the alu element contains a half estrogen-response element/sp1-binding site (alu-ere/sp1), which acts as an in-cis intronic enhancer leading to increased transcription of the progins allele in response to 17beta-estradiol. moreover, alu insertions in the human genome are frequently methylated. some data indicate that the progins-alu does not affect gene transcription due to dna methylation. however, the alu element reduces the stability of the progins transcript compared with the cp allele and does not generate splice variants. the amino acid substitution (v600l) in exon 4 leads to differences in pr phosphorylation and degradation in the two pr variants upon ligand binding, likely because of differences in the three-dimensional structures of the two pr variants. consequently, the pr-l660 (progins) variant displays decreased transactivation activity in a luciferase reporter system and is less efficient in opposing cell proliferation in hamster ovarian cells expressing human pr-a, when compared with the pr-v660 (most common variant). progins variant of pr is less responsive to progestin compared with the most common pr because of reduced amounts of gene transcript and decreased protein activity (66). it has been proven that progins allele has its influence, and represents a risk factor in some patients with breast cancer, endometrial cancer and endometriosis. in pregnancy, we see its influence either in decreased effectiveness of pgrs on p4 or through increased risk for conditions associated with preterm birth. another snp that the authors wanted to emphasize is +331 g/a snp of pr, which is a newly described mutation. its significance lies in its influence on pr-a/pr-b isoform ratio, more specifically, its ability to change the ratio in favor of pr-a. the possible significance of these mutations could be decreased transcriptional regulation of progesterone target genes, which leads to change of pathways and start of preterm birth. several studies have tried to show whether there is an influence of these four snps on modulation of preterm birth. results are biased. some studies (diaz cueto et al., 2008; guoyang et al., 2008; kurtz et al., 2001; oliveira et al., 2011) (67,68,69,70) did not find any connection between polymorphism of pgr and risk of preterm birth. on the other hand, some studies (langmia et al., 2015; ehn et al., 2007; tiwari et al., 2014; mann et al., 2013) (71,72,73,74) have found that mutations of pgrs have a significant influence on modulation of preterm birth. there is a possibility that either mutation in the mother’s or in fetal pgr genes leads to a difference and, consequently, preterm birth. ehn at al. found that mutation in both fetal and maternal prgs contributes to greater possibility of preterm birth. in addition, it has been proven that these mutations of pgr receptors differently influence preterm birth depending on race. this influence is bigger in the africanamerican population. other studies have only confirmed that women with these mutations have a greater risk of preterm birth. progesterone as therapy for prevention of preterm birth supplement of progesterone as prevention of preterm birth appears to be effective, but it has to be kept in mind that it is not an ideal medicine. usage of progesterone as therapy for prevention of preterm birth depends on various factors. first of all, it is important to use the appropriate type of progesterone and to choose appropriate patients. it is also important to use a proper dose, route of delivery and plasma concentration (76,77). the pathway leading to preterm birth is also of importance (78,79). seemedj 2017, vol 1, no. 2 progins mutation of progesterone receptors and its role in premature birth… 63 southeastern european medical journal, 2017; 1(2) studies have shown that clinical efficacy and safety of progesterone therapy can also be altered due to gene polymorphisms of pgrs (75). that fact is especially important in the africanamerican population, where genetic mutations of pgrs occur more often, and lead to resistance to progesterone therapy. there are two major preparations of progesterone used in premature birth therapy/prophylaxis; hydroxyprogesterone caproate, a synthetic progesterone usually used in 250 mg dosage once a week, given as an intramuscular injection; and natural/micronized progesterone in a dosage of 100 mg per day, given vaginally. there is also a rare application of progesterone as vaginal gel in a dosage of 90-200 mg. most common usage of progesterone preparations as prophylaxis of preterm birth starts from 16-20 weeks and lasts until the 36th week of gestation. several meta-analyses and studies have been made about the efficiency of progesterone treatment in preterm birth. table 3. recommendations for progesterone supplementation to prevent preterm birth (85). progesterone preparation dosage and route of administration indications hydroxyprogesterone caproate a 250 mg 250 mg intramuscularly once a week from between 16th and 20th week of gestation until the 36th week of gestation singleton pregnancy, prior spontaneous singleton preterm birth, normal cervix length. singleton pregnancy, prior spontaneous preterm birth of twins, normal cervix length. twins, prior preterm birth. natural progesterone vaginally singleton pregnancy, prior spontaneous singleton preterm birth, normal cervix length. singleton pregnancy, prior spontaneous preterm birth of twins, normal cervix length. twins, prior preterm birth. twins, short cervix. micronized progesterone vaginal gel/vaginal tablet 90 mg /100 mg per day, vaginally singleton pregnancy,prior spontaneous preterm birth of twins, short cervix < 20 mm. progesterone suppository 90-200 mg per day, vaginally, from the moment of diagnosis until the 36th week of gestation singleton pregnancy, prior spontaneous preterm birth of twins, short cervix < 20 mm. seemedj 2017, vol 1, no. 2 progins mutation of progesterone receptors and its role in premature birth… 64 southeastern european medical journal, 2017; 1(2) dodd et al. conducted a meta-analysis of usage of progesterone for prevention of preterm birth in singleton pregnant women with high risk for preterm birth. usage of progesterone made the risk for preterm birth in their current pregnancies lower. (80) meis et al. researched the usage of 17 hydroxyprogesterone caproate in women who had a documented preterm singleton birth in their obstetric anamnesis. therapy started between 16th and 20th week of gestation and was used until the 36th week. prophylaxis reduced the risk of preterm birth in their current pregnancy. (81) da fonseca et al. analyzed the use of progesterone vaginal suppository between 24th and 34th week of gestation. therapy was given to singleton pregnant women with risk factors in their anamneses. risk of preterm birth in their current pregnancies was reduced with prophylaxis. (82) contrary to those findings, opptimum trial, led by norman et al., showed that vaginal progesterone therapy did not reduce fetal and neonatal mortality and morbidity in preterm birth, or preterm birth itself. (83) progress study, led by crowther et al., also did not find any reduction in mortality or morbidity of fetuses or mothers related to preterm birth (84). conclusion in conclusion, usage of progesterone in prophylaxis of preterm birth is still indicated and used worldwide. the most important thing is to choose the right candidates with the right risk factors for preterm birth. recommendations for usage of progesterone for prevention of preterm birth are given in table 3 (85). acknowledgements this manuscript is supported by the faculty of medicine osijek internal scientific project vif2017-mefos-3 “role of progins mutations in progesterone receptors as modulators of risk for premature birth” (project leader jasenka wagner). competing interests: none to declare. references 1. wen sw, smith g, yang q, walker m. epidemiology of preterm birth and neonatal outcome. semin fetal neonatal med 2004;6:429-35. 2. moutquin jm. classification and heterogeneity of preterm birth. bjog int j obst gy 2003;110:30-3. 3. blencowe h, cousens s, oestergaard mz, chou, d, moller ab, narwal r, adler a, garcia cv, rohde s, say l, lawn j. national, regional, and worldwide estimates of preterm birth rates in the year 2010 with time trends since 1990 for selected countries: a systematic analysis and implications. lancet 2012;379:2162-72. 4. anum ea, springel eh, shriver md, strauss jf. genetic contributions to disparities in preterm birth. pediatric res 2009;65:1-9. 5. sheikh ia, ahmad e, jamal ms, rehan m, assidi m, tayubi ia, albasri sf, bajouh os, turki rf, abuzenadah am, damahouri ga, beg ma, al-qahtani m. spontaneous preterm birth and single nucleotide gene polymorphisms: a recent update. bmc genomics 2016;17:759. 6. stanojević m. prevention of preterm birth – neonatologists’ point of view. paediatr croat 2016;60:137-45. 7. romero r, espinoza j, kusanovic j, gotsch f, hassan s, erez o, chaiworapongsa t, mazor m. the preterm parturition syndrome. bjog int j obst gy 2006;113:17-42. 8. gotsch f, romero r, erez o, vaisbuch e, kusanovic jp, mazaki-sovi t, kim sk, hassan s, yeo l. the preterm parturition syndrome and its implications for understanding the biology, risk assessment, diagnosis, treatment and prevention of seemedj 2017, vol 1, no. 2 progins mutation of progesterone receptors and its role in premature birth… 65 southeastern european medical journal, 2017; 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1(2) 85. norowitz er, lockwood cj, barss va. progesterone supplementation to reduce the risk of spontaneous preterm birth. uptodate, topic 16560 version 86.0, https://www.uptodate.com/contents/pro gesterone-supplementation-to-reducethe-risk-of-spontaneous-preterm-birth (acsessed on 16th of december 2017). 86. lockwood cj, kuczynski e. risk stratification and pathological mechanisms in preterm delivery. paediatr perinat epidemiol 2001;15(s2):78-89. seemedj 2020, vol 4, no. 2 newborns after medically assisted fertilization 88 southeastern european medical journal, 2020; 4(2) original article early vital indicators of newborns born after medically assisted reproduction 1 ana-maria milas *1, silvija pušeljić 2,3, jerko arambašić 4, matej šapina 2,5, višnja tomac 2,5 1 institute of maxillofacial and oral surgery, university hospital centre osijek, croatia 2 department of paediatric neurology, genetics, endocrinology and metabolic diseases, paediatric clinic, university hospital centre osijek, croatia 3 josip juraj strossmayer university of osijek, faculty of medicine osijek, croatia 4 institute of cardiology, university hospital centre osijek, croatia 5 paediatric clinic, university hospital centre osijek, croatia *corresponding author: ana-maria milas, milas.anam@gmail.com received: feb 29, 2020; revised version accepted: sep 28, 2020; published: nov 12, 2020 keywords: high-risk pregnancies, in vitro fertilization, natural conception, prematurity, low birth weight abstract aim: in this case-control study we wanted to compare the perinatal outcome of naturally conceived newborns to those born after in vitro fertilization (ivf). methods: at the university hospital centre osijek, in the period from january 2014 to december 2016, we analysed 120 in vitro fertilization pregnancies and compared to 120 natural conception pregnancies. characteristics of mothers, birth method, perinatal outcome, and vital characteristics of newborns were analysed. results: ivf-et pregnancies included 70.0% singleton pregnancies, 28.3% twin and 1.7% triplet pregnancies, while all naturally conceived pregnancies were singletons. when pregnancies were compared between ivf-et and naturally conceived groups, the following characteristics of mothers were established: age 34.83 (5.8):30.39 (3.9); previous pregnancy 13.3%:56.7%; complications in pregnancy 50.0%:25.0%; caesarean section (cs) 69.2%:35.8%. all of these were statistically significant (p < 0.001). comparison of newborns between those two groups established the following: prematurity 39.5% vs. 12.7%; lowest bw 2,114 vs. 3,000 grams; lowest ga 22.29 vs. 28.71 weeks. in the ivf-et group, 5.7% of newborns had a gestational age of 22-25 weeks, but there were no newborns of that gestational age among the naturally conceived newborns. these were also statistically significant differences (p < 0.001). apgar score was 10 for both groups, but the difference was in the interquartile range, the values of which were lower in the ivf-et group. conclusion: in vitro fertilization pregnancies are high risk due to the characteristics of both the mother and the infant and, as such, require special attention and care. (milas am, pušeljić s, arambašić j, šapina m, tomac v. early vital indicators of newborns born after medically assisted reproduction. seemedj 2020; 4(2); 88-96) seemedj 2020, vol 4, no. 2 newborns after medically assisted fertilization 89 southeastern european medical journal, 2020; 4(2) introduction the first baby girl born through medically assisted fertilization (maf) was born back in 1978, and nowadays children that have been born through implementation of this technique make up 1.7% to 4.0% of the population in developed countries [1, 2]. there are only a few fields in medicine that have progressed as rapidly as maf, which, today, represents the last resort for infertile couples. clinical guidelines indicate that the availability of a high-quality in vitro fertilization (ivf) and embryo transfer (et) programme can significantly contribute to the birth rate [3]. after four decades of experience in the field of maf, there are now relevant studies being conducted in china, australia, great britain and belgium, which clearly show that children born after maf differ from naturally conceived children [4-7] in terms of perinatal outcome and vital characteristics. although available studies differ in methodology, they all include a comparison of pregnancies conceived after maf (number of foetuses, complications in pregnancy) and of perinatal outcomes (gestational age, birth weight) after naturally conceived pregnancies [4-11]. those studies have shown that newborns born after ivf-et are more sensitive during the perinatal period in comparison to newborns born after naturally conceived pregnancies. such results are to be expected because the characteristics of women who undergo maf differ from those of women from the general population (higher age, lower parity, comorbidity, infertility or subfertility). it has been demonstrated that maf methods have contributed to a greater number of multiple pregnancies, primarily twin pregnancies, but also triplet and quadruplet pregnancies [13]. studies conducted in croatia are consistent with the results of the above-mentioned research, and they likewise show that there are significantly more multiple pregnancies in the ivf group than with naturally conceived pregnancies [8, 14]. newborns from the ivf group are born with lower birth weight (bw) and gestational age (ga), and they stay in the intensive care unit (icu) longer after birth. by researching available literature, we observed that there is a 30-40% higher risk of congenital malformations after the ivf-et procedure, and this population had a higher incidence of perinatal mortality [5, 8, 10]. perinatal outcomes of newborns born after maf also vary in regard to the different methods of maf. a pilot study conducted in vienna from 2003 to 2009 in a tertiary health care centre showed a poorer perinatal outcome (lower bw, lower ga, lower apgar score) among children who were conceived using the icsi method, compared to children who were conceived after ivf-et treatment [15]. the association of ivf with neurodevelopmental disorders has been demonstrated in numerous published studies, which showed that children born after ivf have an increased risk of developing cerebral palsy, as well as various disorders resulting from erroneous genomic imprinting [4, 5, 16]. in exploring the relationship between ivf and autism spectrum disorders (asd), we found conflicting studies. a swedish study, which was conducted over a period of 25 years, showed that the ivf procedure was not associated with a higher risk of asd, but with a significantly higher risk of developing mental retardation. on the other hand, meta-analysis by liu et al. showed that maf was associated with a higher percentage of asd [17, 18]. in accordance with these findings, it is justified to claim that the perinatal outcome of ivf cannot be equal to the outcome after natural conception, bearing in mind the diseases and conditions which led to the need for assisted fertilization; likewise, pregnancies after maf are high-risk pregnancies, whether they are singleton or multiple. the aim of the study was thus to examine the difference in the vital characteristics of newborns born after medically assisted fertilization in comparison with natural conception, as well as characteristics and the course of pregnancy in women who conceived through medically assisted fertilization and those who conceived naturally. seemedj 2020, vol 4, no. 2 newborns after medically assisted fertilization 90 southeastern european medical journal, 2020; 4(2) material and methods the study was designed as a three-year retrospective case-control study and was conducted in the gynaecology clinic and neonatal intensive care unit (nicu) of the university hospital centre osijek. the study included all infants who were born from pregnancies through assisted reproduction and the first following infant born after natural conception at the university hospital centre osijek from 1 january 2014 to 31 december 2016. ethical approval for this study was given by the ethics committee of the faculty of medicine. general data on the mother (age, occupation, marital status), parity data (number of births, number of miscarriages, accompanying comorbidities, pregnancy and comorbidities during pregnancy) and data on the characteristics of newborns (sex, length, apgar score) were collected. statistical analysis the data were analysed using statistical procedures for testing differences and correlations, using the statistical program spss 17.0. the mean values of the continuous variables are expressed by the arithmetic mean and standard deviation for normally distributed variables and the median and interquartile range for variables that are not normally distributed. the mann-whitney u test for nonparametric analysis was used to determine the differences between the two independent samples. the χ2 test was used to determine the differences between the proportions between the two independent samples. statistical significance was accepted for p < 0.05. results a total of 240 births were analysed, of which 120 were pregnancies after ivf-et, while the control group included pregnancies where birth occurred immediately after the observed ivf birth, likewise a total of 120 births. since there were multidisciplinary pregnancies among the observed pregnancies, a total of 277 subjects were included in the study. a total of 240 mothers was divided into two groups – the control group, or 120 mothers that conceived naturally, and the ivf group, or 120 mothers that conceived after ivf-et treatment. mothers in the ivf group are older than those in the control group (arithmetic mean ((sd) of age 34.83 (5.8) vs. 30.39 (3.9)), p < 0.001. according to the level of education, mothers from the ivf group have a higher level of education (academic degree), while mothers from the control group predominantly have a low or medium level of education (secondary school degree, primary school or no education), which is a statistically significant difference, p < 0.001 (figure 1). figure 1. level of education of mothers control group n = 120 (%) ivf group n = 120 (%) p* (χ2 test) level of educatio n high or higher (academic degree) 29 (24.2) 66 (55.0) < 0.001 low or medium (secondary school, primary school, no education) 91 (75.8) 54 (45.0) seemedj 2020, vol 4, no. 2 newborns after medically assisted fertilization 91 southeastern european medical journal, 2020; 4(2) figure 2. previous pregnancies and miscarriages by groups ivf n = 120 (%) control group n = 120 (%) p* (χ2 test) pregnancies yes 16 (13.3) 68 (56.7) < 0.001 no 104 (86.7) 52 (43.3) miscarriages no 95 (79.2) 96 (80.0) > 0.999 yes 25 (20.8) 24 (20.0) a significantly higher portion of mothers in the ivf group had not been pregnant before (p < 0.001; *χ2 test), as shown in figure 2. comparison of the previous number of miscarriages by group did not show a statistically significant difference between the groups, χ2 test (p > 0.999) (figure 2). of the total number of mothers (240), 150 (62.5%) mothers experienced no complications during pregnancy (hellp syndrome, bleeding, use of tocolytics, etc.). however, group distribution shows that more complications during pregnancy were experienced in the ivf group (60% of mothers with complications are from the ivf group). statistically significant differences were found for the groups in regard to the method of delivery, χ2 test (p < 0.001). vaginal delivery was the predominant way of giving birth within the control group, whereas pregnancies within the ivf group were mostly completed by caesarean section, as shown in figure 3. the highest portion of pregnancies in this study were singleton pregnancies, followed by twin pregnancies. triplets were found in two pregnancies. all multiple pregnancies were from the ivf group, as shown in figure 4. figure 3. method of delivery ivf group n = 120 (%) control group n = 120 (%) p* (χ2 test) vaginal 37 (30.8%) 77 (64.2%) < 0.001 cs 83 (69.2%) 43 (35.8%) figure 4. distribution by number of offspring between groups ivf group n = 120 control group n = 120 p* (χ2 test) singletons 84 (70.0%) 120 (100%) < 0.001 twins 34 (28.3%) 0 (0.0%) triplets 2 (1.7%) 0 (0.0%) seemedj 2020, vol 4, no. 2 newborns after medically assisted fertilization 92 southeastern european medical journal, 2020; 4(2) when comparing these two groups based on the sex of the newborn, there were no statistically significant differences. statistically significant differences were found for every numerical variable (birth weight, gestational age, apgar score) except for the mean value of ph (p = 0.747). mean gestational age in the ivf group was 35.57 weeks (22.29 – 37.57), and it was 38.14 (28.71 – 39.29) weeks in the control group. the shortest duration of pregnancy in the control group was 28.71 weeks, and it was 22.29 weeks in the ivf group (figure 5). the distribution of newborns by gestational age within the groups shpwed that two children were born between the 22nd and 25th week of pregnancy in the ivf group (p < 0.001; fisher’s exact test), while there were no births within that period in the control group (figure 6). figure 5. duration of pregnancy (in weeks) number of mothers arithmetic mean (sd) minimum maximum control group 120 38.92 (2.06) 28.71 42.14 ivf group 120 36.22 (4.39) 22.29 40.86 (data are shown as arithmetic mean and standard deviation (sd)) figure 6. distribution of newborns by gestational age gestational age (weeks) ivf group number (%) control group number (%) 22-25 9 (5.7) 0 (0.0) 26-29 10 (6.4) 1 (0.8) 30-33 12 (7.6) 3 (2.5) 34-36 31 (19.7) 10 (8.4) 37+ 95 (60.5) 105 (88.2) there were 12% of births in control group which were premature (n =12), while 39% of total births in ivf group were preterm births (n = 62). the high proportion of multiple pregnancies within the ivf group resulted in a significantly higher percentage of prematurity in that group, (p < 0.001; χ2 test, †mann-whitney u test, data not shown). likewise, the birth weight of the newborns in the ivf group was lower than of those in the control group, as was expected. the lowest birth weight in the ivf group was 2114 grams, and the highest was 3330 grams. in the control group, the lowest birth weight was 3000 grams, and the highest 3785 grams; these values were significantly different between the groups. in spite of the same median of the average apgar score, which equals 10 in both groups, the difference is in the interquartile range, which is shifted to lower values (9.5-10) in the ivf group (data not shown). seemedj 2020, vol 4, no. 2 newborns after medically assisted fertilization 93 southeastern european medical journal, 2020; 4(2) figure 7. birth weight distribution of newborns median (25.00-75.00) ivf group (range) control group (range) birth weight (grams) 2880 (2114 3330) 3350 (3000 3785) discussion research regarding the vital characteristics of newborns born after in vitro fertilization has not been conducted so far at the university hospital centre osijek, even though medically assisted fertilization has been performed in this institution in accordance with the highest standards for over a decade. since our search of the literature did not produce any studies that compare the socioeconomic characteristics of the mother, such as the mother’s level of education and marital status, we included these parameters in our study. the results were as expected: there is a significantly higher percentage (55.0%) of mothers with a high or higher level of education in the ivf group, while in the control group this percentage is 24.20%. such a result can be explained by an increase in the number of women in the academic community, their commitment to their careers and professional development and, consequently, the postponed maternity. this result can also be directly related to the mothers’ age – the average age of ivf mothers was 34.88, while it was 30.39 in the control group. this result is consistent with the result obtained in a study that included the same parameter, but which also linked singleton pregnancy with higher maternal age, which was not analysed in our study [19]. by observing the mothers’ marital status, we found that there is a higher percentage of mothers who are married that conceived by ivf, compared to the control group. this result did not surprise us, given that the goal of most married couples is to have children, which means that they have to treat infertility if it exists. in the ivf group, 110 (91.70%) mothers were married, while 97 (80.80%) mothers were married in the control group. as expected, the ivf group contains 104 (86.7%) mothers that had not been pregnant previously, which is statistically significant when compared to the control group, where the observed pregnancy was the first pregnancy for 52 (43.30%) mothers. this brings us back to the definition of medically assisted reproduction, which states that this method is the last resort for treating infertility [3]. even though we could assume that mothers from the ivf group have a higher incidence of miscarriages, there is no statistically significant difference in this parameter between the two groups. out of a total of 240 respondents, 150 of them (62.50%) experienced no complications during pregnancy, but when analysed each group individually, 60 (50.0%) of the ivf mothers experienced complications during pregnancy, while that number was 30 (25.0%) in the control group. this result is not surprising, since the mothers who conceived through ivf could not conceive naturally, which is also why they have a more complicated pregnancy. out of the total number of pregnancies (240), 126 (52.50%) were finished with the caesarean section. if we divide that number according to group, we get a statistically significant difference (p < 0.001). 69.2% of caesarean sections were performed in the ivf group, while vaginal birth occurred in 30.80% of women. in contrast, in the control group, 35.80% of pregnancies were completed with the caesarean section and 64.20% of women experienced vaginal birth. the obtained number can be directly related to the number of newborns, where we also found a statistically seemedj 2020, vol 4, no. 2 newborns after medically assisted fertilization 94 southeastern european medical journal, 2020; 4(2) significant difference (p < 0.001). out of 120 pregnancies in each group, 28.30% of them in the ivf group were twin pregnancies, while 1.70% of them were triplet pregnancies. in the control group, all pregnancies were singleton pregnancies. the fact that all multiple pregnancies were in the ivf group justifies the prevalence of caesarean section as the method of delivery, since multiple pregnancy is one of the indications for caesarean section. our results from this study are similar to those obtained in studies that also compared these parameters [8, 19]. taking into account the data from the previous section, shorter duration of pregnancies in the ivf group in comparison with the control group is justified. the shortest pregnancy in the ivf group lasted 22.29 weeks, while the shortest one in the control group lasted 28.71 weeks. if we put those numbers in the context of perinatal outcomes, we get a statistically significant difference for a newborn child. there is a higher incidence of premature infants in the ivf group than in the general population. when duration of pregnancies was expressed in weeks and analysed both groups, one could observe that there are no newborns born between the 22nd and 25th week in the control group, and only one newborn born between the 26th and 29th week. most children are born “at full term”, i.e. 88.20% from the 37th week onwards. in the ivf group, the situation is very different. 5.70% (9) of the newborns were born in the critical period between 22 and 25 weeks, and “only” 60.50% from the 37th week onwards. it is clear that all of the above parameters also affect the birth characteristics of newborns in both groups. median birth weight in the control group was 3,350 grams, with a higher lowest birth weight than the one in the ivf group. in the ivf group, median birth weight was 2,880 grams, with the very low 2,114 grams as the lowest value. the highest birth weight in the ivf group was lower than the average birth weight in the control group, which is not significantly different from data in other studies [8, 19, 20]. conclusion with the direct link between medically assisted reproduction and low birth weight, as with gestational age in this study and high maternal age, we can conclude that ivf-conceived pregnancies are high-risk pregnancies and should as such receive special attention and care. since medically assisted reproduction is more common now than it was before, it is the right time to see early vital indicators and collect them to compare with later data. this study thus opens a lot of possibilities and indicates the need for long-term prospective follow-up of these respondents, in order to obtain more information and findings about the outcomes of children born after medically assisted fertilization. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. kamel rm. assisted reproductive technology after the birth of louise brown. j reprod infertil. 2013; 14(3):96–109. 2. zhu l, zhang y, liu y, zhang r, wu y, huang y, liu f, li m, sun s, xing l, zhu y, chen y, xu l, zhou l, huang h, zhang d. maternal and live-birth outcomes of pregnancies following assisted reproductive technology: a retrospective cohort study. sci rep 2016; 6:35141. 3. šimunić v. kliničke smjernice za medicinski potpomognutu oplodnju. medix.2013;104/105: 132-146. 4. yue-hong l, wang n, jin f. long-term follow-up of children conceived through assisted reproductive technology. j zhejiang univ sci b. 2013; 14(5):359-371. 5. british fertility society. in vitro fertilisation: perinatal risk and early childhood outcomes. scientific impact paper no. 8. 2012; 2-12 seemedj 2020, vol 4, no. 2 newborns after medically assisted fertilization 95 southeastern european medical journal, 2020; 4(2) 6. wang ya, sullivan ea, black d, dean j, bryant j, chapman m. preterm birth and low birth weight after assisted reproductive technology-related pregnancy in australia between 1996-2000. fertil steril. 2005; 83(6):1650-8. 7. ombelet w, martens g, bruckers l. pregnant after assisted reproduction: a risk pregnancy is born! 18-years perinatal outcome results from a population-based registry in flanders, belgium. facts view vis obgyn. 2016; 8(4):193-204. 8. peter b. bilić i. prpić i. perinatalni i neuromotorni ishod novorođenčadi rođenih iz trudnoće nakon postupka ivf et et. medicina 2007; 43:270-278. 9. cavoretto p, candiani m, giorgione v, inversetti a, abu-saba mm, tibero f, sigismondi c, farina a. risk of spontaneous preterm birth in singleton pregnancies conceived after ivf/icsi treatment: metaanalysis of cohort studies. ultrasound obstet gynecol. 2018; 51(1):43-53. 10. padney s, shetty a, hamilton m, bhattacharya s, maheshwari a. obstetric and perinatal outcomes in singleton pregnancies resulting from ivf/icsi: a systematic review and meta-analysis. hum reprod update.2012; 18(5):485-503. 11. quin jb, sheng xq, wu d, gao sy, you yp, yang tb, wang hb. worldwide prevalence of adverse pregnancy outcomes among singleton pregnancies after in vitro fertilization/intracytoplasmic sperm injection: a systematic review and metaanalysis. arch gynecol obstet. 2017; 295(2):285-301. 12. stojnic j, radunovic n, jeremic k, kotlica bk, mitrovic m, tulic i. perinatal outcome of singleton pregnancies following in vitro fertilization. clin exp obstet gynecol. 2013; 40(2):277-283. i author contribution. acquisition of data: milas am, pušeljić s, arambašić j, tomac v administrative, technical or logistic support: milas am, pušeljić s, arambašić j, tomac v analysis and interpretation of data: milas am, pušeljić s, arambašić j, šapina m, tomac v 13. andersen-nyboe a, goossens v, ginaroli l, felberbaum r, de mouzon j, nygren kg. assisted reproductive technology in europe, 2007. results generated from european registers by eshre. hum reprod. 2012; 27(4):954–966. 14. tomić v. tomić j. neonatal outcome of ivf singletons versus naturally conceived in women aged 35 years and over. arch gynecol obstet. 2011; 284:1411–1416. 15. nouri k. ott j. stoegbauer l. pietrowski d. frantal s. walch k. obstetric and perinatal outcomes in ivf versus icsi-conceived pregnancies at a tertiary care center – a pilot study. reprod biol endocrinol. 2013; 11:84. doi: 10.1186/1477-7827-11-84 16. zhu jl, hvidtjorn d, basso o, obel c, thorsen p, uldal p, olsen j. parental infertility and cerebral palsy in children. hum reprod. 2010; 25(12):3142-5. 17. sandin s, nygren kg, iliadou a, hultman cm, reichenberg a. autism and mental retardation among offspring born after in vitro fertilization. jama. 2013; 310(1):75-84. 18. liu l, gao j, he x, cai y, wang l, fan x. association between assisted reproductive technology and the risk of autism spectrum disorders in the offspring: a meta-analysis. sci rep. 2017; 7:46207. 19. vulić m, roje d, meštrović z, strinić t, stipić i, vrkić i. is there difference in perinatal outcome of singleton and twin pregnancies after assisted conception: two-year experience. acta clin croat 2013; 52:241-246. 20. kasdallah n, ben salem h, kbaier h, bouguerra c, blibech s, douagi m. premature birth, low birth weight and birth defects after assisted reproductive therapies. a 18-year comparative study. tunis med. 2017; 95 (2):103-108.. i conception and design: milas am, pušeljić s, arambašić j, šapina m, tomac v critical revision of the article for important intellectual content: milas am, pušeljić s, arambašić j, šapina m, tomac v drafting of the article final approval of the article seemedj 2020, vol 4, no. 2 newborns after medically assisted fertilization 96 southeastern european medical journal, 2020; 4(2) guarantor of the study: milas am, pušeljić s, arambašić j, šapina m, tomac v obtaining funding: milas am, pušeljić s, arambašić j, šapina m, tomac v provision of study materials or patients: milas am, pušeljić s, arambašić j, tomac v statistical expertise: milas am, pušeljić s, arambašić j, šapina m, tomac v other: milas am, pušeljić s, arambašić j, šapina m, tomac v seehsj south eastern europe health sciences journal southeastern european medical journal seemedj faculty of medicine osijek josip juraj strossmayer university of osijek j. huttlera 4 croatia – 31000 osijek seemedj@mefos.unios.hr seemedj.mefos.unios.hr editorial dear colleagues, i am happy to present you with the new issue of southeastern european medical journal (seemedj 2021; 5(1):1-190). this is the ninth number in the fifth year of journal publication. this issue brings papers from authors from different countries in south-eastern europe and worldwide – several topics of general interest. in the first part of the journal one can read articles on epidemiology and public health issues related to infectious diseases. in the era of covid-19 pandemic it is easy to forget that many other contagious diseases present an enormous burden to health systems. two articles from west africa (ayenigbara et al, and olofintuyi et al, nigeria) point towards the necessity to prevent or contain diseases such as lassa fever and under-five infectious diarrhea, respectively, both diseases having high morbidity and mortality, while is possible to combat both with directed public health care strategies. in europe, newly emerging issue is minor outbreaks of diseases that have been successfully eliminated and became sporadic due to vaccination programs, such as measles (article by borocz et al, hu). authors pointed out that suspended immunization activities due to the covid-19 surge might be an ominous precursor to a measles resurgence. finally, cytomegalovirus (cmv) infection is common opportunistic infections in kidney transplant recipients, and šisl & zibar (cro) provide suggestions for preemptive. cmv prophylaxis and cmv-dna testing in this group of patients. covid-19 pandemic also revealed necessity to further study thrombin homeostasis, since a number of data suggests a crucial role of thrombin in different pathologies accompanied by blood coagulation disorders, in particular diseases causing endothelial dysfunction (korolova, ukr). on the other side, chronic, non-contagious diseases, such as diabetes mellitus, pose challenging questions, as well. bardak et al (cro) and mehovic et al (bih) investigate diabetic treatments relation to diabetic retinopathy, and quality of life of diabetic patients with cardiovascular complications, respectively. the need to have important psychometric instrument in croatian language is fulfilled by translation of clance impostor phenomenon scale from english to croatian by čarapina-zovko et al (cro). other published articles are concerned with topics of robotic-assisted rehabilitation of neurological patients (blazincic et southeastern european medical journal seemedj faculty of medicine osijek josip juraj strossmayer university of osijek j. huttlera 4 croatia – 31000 osijek seemedj@mefos.unios.hr seemedj.mefos.unios.hr al, cro), and with often overlooked condition underactive bladder syndrome (radoja, cro). kovačević et al (cro) evaluated immunophenotypes and proliferation index in regard to axillary lymph node invasion in breast cancer in monocentric cross-sectional study. importance of telemedicine, that we are aware of even more nowadays, in necessity to keep the physical distance, is discussed in the article by mihalj et al (cro); and application of telemedicine in otorhinolaryngology, with historical overview, current status and future perspective is presented, two papers (juzbašič – on oral hygiene in disabled persons and stanić on dementia and life habits, cro) are co-edited by senior lecturer mirna sabljar, phd, from the faculty of art and culture of university of osijek, as contributions from their international meeting on art and disabilities, giving to present issue of seemedj interdisciplinary perspective. oral health and dental occlusion in young adults as well is a point of research in article by (strikić đula et al, cro) and body compartmentalization in athletes and non-athletes in article by seper & nesic, (cro). finally, always intriguing problem of abortion, legislative and practice is a topic of review by negro et al (it). it is important to mention the art work at the cover page of this issue. it is a painting of strossmayer’s park in đakovo, bellowed part of the city to all inhabitants, painted by arpad franjo mesaroš, from the museum of fine arts in osijek (selected by ms valentina radoš, senior curator). arpad franjo mesaroš was a painter of personal and solitary art, renowned in croatia and born in slavonija county. on a behalf of editorial board and my own, i warmly greet our readers and invite them to join us in the endeavor of publishing own scientific work in seemedj. sincerely, ines drenjančević, md, phd editor-in-chief southeastern european medical journal (seemedj) seemedj 2021, vol 5, no. 1 cytomegalovirus in kidney transplantion 17 southeastern european medical journal, 2021; 5(1) original article cytomegalovirus infection in kidney transplant recipients 1 dino šisl 1*, lada zibar 2 1 university of zagreb, school of medicine, department of physiology and immunology, croatian institute for brain research, laboratory for molecular immunology, zagreb, croatia 2 department of nephrology, internal medicine clinic, merkur clinical hospital, zagreb, croatia; department of pathophysiology, faculty of medicine, josip juraj strossmayer university osijek, croatia *corresponding author: dino šisl, dino.sisl@mef.hr received: feb 1, 2021; revised version accepted: mar 15 2021; published: apr 28, 2021 keywords: cytomegalovirus, kidney transplantation, cmv-dna, prophylaxis, valganciclovir abstract introduction: present study examined the frequency of cmv infection during follow-up using quantitative nucleic acid amplification testing, the frequency of administration of infection prophylaxis, viremia and infection in kidney transplant recipients who underwent transplantation (tx) at the university hospital center osijek. materials and methods: 107 kidney recipients who underwent transplantation in the period 20 october 2007 – 24 august 2016 were included. demographic and clinical data, data about pretransplantation cmv igg test results of recipients and their donors, data about cmv prophylaxis, viremia, infection, and kidney transplant function were taken from medical records and analyzed. results: 92.5% of kidney recipients and 86% of donors were cmv igg positive before tx. 28% of recipients were cmv-dna positive at some point after tx, none of whom received a transplant from an igg negative donor. 89.7% of participants received cmv prophylaxis. seven participants developed cmv disease, 2 of whom were not administered prophylaxis. participants were tested for cmv-dna once a year (median; min 0 max 6). cmv disease was marginally more frequent in those who did not receive valganciclovir prophylaxis (p = 0.066). conclusion: it seems wise to enforce the administration of cmv prophylaxis and cmv-dna testing in accordance with protocol, in order to detect viremia on time and to implement preemptive treatment, aiming at prevention of clinical manifestation of infection and preservation of graft function. (šisl d, zibar l. cytomegalovirus infection in kidney transplant recipients. seemedj 2021; 5(1); 17-24) seemedj 2021, vol 5, no. 1 cytomegalovirus in kidney transplantion 18 southeastern european medical journal, 2021; 5(1) introduction cytomegalovirus (cmv) infection is one of the most common opportunistic infections in kidney transplant recipients. there are two ways of acquiring the infection: reactivation of the virus or de novo infection of an immunocompromised patient. the infection can be asymptomatic, it can jeopardize the function of the graft and/or cause a systemic infection and even death. cmv infection is defined as virus isolation or detection of viral proteins (antigens) or nucleic acid in any bodily fluid or tissue specimen, regardless of symptoms. cmv disease is defined as evidence of cmv infection with attributable symptoms and can be further categorized as a viral syndrome (i.e. fever, malaise, leukopenia, and/or thrombocytopenia), or as a tissueinvasive (“end organ”) disease. in recent years, administration of cmv infection prophylaxis during the first post-transplantation months has become a standard part of therapy after kidney transplantation (tx). it is also recommended to observe cmv viremia using quantitative nucleic acid amplification testing (qnat) that detects cmv-dna (deoxyribonucleic acid). prophylaxis and diagnostics raise the cost of treatment. however, modern protocols justify this approach with the lower total cost of the treatment, since it is deemed that this approach leads to fewer complications. the prophylaxis and diagnostics protocol at the university hospital center (uhc) osijek is not strict and has changed in recent years, while the approach to prophylaxis and diagnostics is not completely uniform. the aim of the study was to examine the frequency of cmv infection during follow-up using quantitative nucleic acid amplification testing (qnat) for cmv-dna, the frequency of administration of cmv infection prophylaxis, and the frequency of cmv viremia and infection in kidney transplant recipients who received their transplant in uhc osijek during the period between 20 october 2007 and 24 august 2016. materials and methods the study included 107 participants, 60 men and 47 women, who underwent kidney tx in uhc osijek in the period between 20 october 2007 and 24 august 2016. median age at the time of the tx was 51 (min. 27, max. 71), while at the time of the study it was 57 (min. 32, max. 74). research methods included collecting data from medical records and statistical analysis. the following data were analyzed: demographic data of the participants (age, sex), clinical features of the participants (primary kidney disease, data about dialysis and tx), data about cmv igg test results of both recipients and their donors prior to tx, data about cmv prophylaxis, viremia, infection, and about kidney transplant function. statistical analysis data analysis was conducted using spss for windows (version 16.0, spss inc., chicago, il, usa). nominal data were expressed as absolute and relative frequencies. numerical data were expressed as mean and standard deviation (sd) in case of normal distribution, and as median and range (min. – max. or interquartile range, iqr) in case of asymmetric distribution. the kolmogorov-smirnov test was used to analyze the normality of distribution of the variables. differences in frequencies were tested using the chi-square test, in numeric variables of normal distribution using student’s t-test and mannwhitney u test by asymmetric distribution. correlations between numeric variables were tested using spearman’s rho test. statistical significance was accepted at p < 0.05. results for 97 kidney recipients (90.7%) this was the first tx, for 7 of them (6.5%) the second, and for 3 of them (2.8%) the third one. table 1 shows the demographic data of the participants. female participants were significantly older both at the time of the study (mann-whitney u test, p = 0.003) and at the time of the tx (mann-whitney u test, p = 0.001). by september 2016, 12 (11.2%) participants, 6 men and 6 women, had died, one of whom was cmv igg negative before the tx. seemedj 2021, vol 5, no. 1 cytomegalovirus in kidney transplantion 19 southeastern european medical journal, 2021; 5(1) there was no significant difference in mortality between men and women (mann-whitney u test, p = 0.654). table 1. demographic data of patients number of patients (%) male female 60 (56%) 47 (44%) age (median in years) at the time of the study (iqr) 53 (45–59) 60 (53–65) at the time of the tx* (iqr) 50 (42–54) 55 (49–61) *transplantation table 2. cmv* serology data n % cmv igg prior to tx recipient positive 99 92.5 negative 8 7.5 donor** positive 92 86 negative 14 13.1 recipient–donor pairs based on cmv serology r+/d+ 84 79.2 r+/d14 13.3 r-/d+ 8 7.5 r-/d0 0 *cytomegalovirus, **data for one donor were unavailable r – recipient, d – donor table 2 shows the cmv serology data. protocolar administration of cmv prophylaxis was introduced in february 2009. table 3 shows data related to prophylaxis administration and development of cmv infection and disease. two out of 3 (66.7%) participants who were not administered prophylaxis and 5 out of 27 (18.5%) of those who were administered prophylaxis developed a clinical manifestation of disease, with no significant (rather, marginal) difference in occurrence (mann-whitney u test, p = 0.066). sixteen (53.4%) positive participants were men, while 14 (46.6%) were women. no significant difference was found in the frequency of cmvdna positivity between the sexes (mannwhitney u test, p = 0.671). . seemedj 2021, vol 5, no. 1 cytomegalovirus in kidney transplantion 20 southeastern european medical journal, 2021; 5(1) table 3. cmv* prophylaxis and infection data n % prophylaxis administered 96 89.7 not administered 11 10.3 duration (mean, in months + sd) 4 ± 2 cmv infection and disease positive for cmv-dna** total 30 28 received prophylaxis 27 90 did not receive prophylaxis 3 10 cmv igg*** positive prior to tx 26 86.7 cmv igg negative prior to tx 4 13.3 r+/d+ 26 86.7 r-/d+ 4 13.3 cmv disease total 7 10 received prophylaxis 5 71.4 did not receive prophylaxis 2 28.6 *cytomegalovirus, **positive for cmv-dna was defined as having > 1000 copies/ml, ***immunoglobulin g, r – recipient, d – donor the group in which the recipient was cmv igg positive and the donor negative was significantly less often cmv-dna positive at some point after the tx in comparison with the group in which the recipient was cmv igg negative and the donor positive (mann-whitney u test, p = 0.002), and in comparison with the group in which both were cmv igg positive (mann-whitney u test, p = 0.016). there was no significant difference in the frequency of positivity at some point after the tx between the group in which both were positive and the one in which the recipient was cmv igg negative and the donor positive (mann-whitney u test, p = 0.159). no recipient of a cmv igg negative donor became cmv-dna positive. there was no pairing in which both the recipient and the donor were cmv igg negative. table 4 shows data related to cmv-dna testing and follow-up. according to the kidney function criteria for prophylaxis dosing, the participants received the appropriate drug dose. table 5 shows graft function data. there was no significant difference between the sexes in creatinine concentration in the serum at the start of administration of prophylaxis (mann-whitney u test, p = 0.365). the group in which the recipient was cmv igg positive and the donor negative had significantly lower serum creatinine concentration at the end of prophylaxis administration compared to the group in which both were cmv igg positive (mann-whitney u test, p = 0.009). women had significantly lower serum creatinine concentration than men at the end of prophylaxis (mann-whitney u test, p = 0.002). seven out of 30 participants (23.3%) who were cmv-dna positive at some point after the tx developed cmv disease. in 2 of them, it manifested as invasive cmv disease, while in the other 5 it manifested as a viral syndrome with leukopenia, fever, malaise, loss of appetite, diarrhea, and weight loss. seemedj 2021, vol 5, no. 1 cytomegalovirus in kidney transplantion 21 southeastern european medical journal, 2021; 5(1) table 4. cmv-dna* testing and follow-up data n number of tests performed total 442 per patient (median + iqr) 4 (2–6) per patient year 1.03 follow up duration patient years 426 median + iqr 4 (2–6) min – max 0–9 *cytomegalovirus deoxyribonucleic acid table 5. graft function data value gfr* (in ml/min/1.73 m²) start of prophylaxis – median (iqr) 35 (19.5–48.5) n % value > 60 14 14.6 40–59 26 27.1 25–39 23 24 10–24 24 25 < 10 6 6.3 unavailable 3 3.1 median (iqr) creatinine (µmol/l) start of prophylaxis men 187 (127–264) women 152 (119–262) end of prophylaxis men 129 (111–145) women 103 (93–141) r+/d+ 128 (101–148) r+/d106 (94–110 ) r-/d+ 119 (99–166 ) *glomerular filtration rate, r – recipient, d – donor discussion kidney tx has been performed at the uhc osijek since 2007, and the availability of cmvdna diagnostics dates back to 2009. before 2007, patients treated at the uhc osijek dialysis department had their tx at uhc zagreb, uhc rijeka or merkur clinical hospital, and cmv diagnostics was performed in zagreb or rijeka. this study included only the patients whose tx and diagnostics were performed in osijek. cmv is widely present in the population, which has been shown in numerous studies, according to which the prevalence of seropositivity to cmv igg ranges from 30 to 97% (1, 2). results of this study agree with those results, finding that 92.5% of recipients and 86% of their donors were cmv igg positive prior to the tx. this could be considered as a high prevalence. since cmv presents a significant risk of morbidity and mortality in the population of persons who have received transplants, the importance of prevention of reactivation or de novo infection has been recognized, whether it is performed by administering prophylaxis or through preemptive treatment (3 – 7, 14). prevention of cmv infection at the uhc osijek started in february 2009 with administration of oral valganciclovir, at first during 3 months for cmv igg positive and 6 months for cmv igg negative recipients, and since october 2014, a universal 6-month prophylaxis has been in use. seemedj 2021, vol 5, no. 1 cytomegalovirus in kidney transplantion 22 southeastern european medical journal, 2021; 5(1) taking that into consideration, 96 of 107 kidney recipients, or 89.7%, were administered prophylaxis, which is a large share of recipients. the standard dose of oral valganciclovir in cmv prophylaxis is 900 mg per day. that dose is adjusted for renal function, an indicator of which is creatinine clearance (or calculated gfr). dose adjustment is extremely important since valganciclovir can cause nephrotoxicity and thus jeopardize the graft function. some of the side effects of valganciclovir, such as leukopenia, nausea, diarrhea, and elevated serum liver enzymes, overlap with the symptoms of cmv infection and disease, which is why finding the actual cause of such symptoms is of great importance. regular testing for cmv-dna plays an important role, and it can help with early detection of infection and administration of preemptive treatment already at low viral loads, which could lead to prevention of clinical manifestations and better preservation of graft function, since preemptive treatment has been linked to fewer toxic effects on the transplanted kidney in comparison with the prophylaxis (8). cmv infection and disease can, however, appear despite preventive therapy, and the reasons for this are incorrect dosing, discontinuation of preventive therapy, or simply a failure of such therapy (4). thirty recipients, or 28% of those who were included in the study, developed cmv infection (and 7 of them had cmv disease), which is a relatively high number if we consider the fact that most of them took prophylaxis. a primary risk factor for the development of cmv viremia and disease is considered to be the serostatus of the recipient–donor pair regarding cmv igg antibodies; the pairs in which both are positive and in which the recipient is negative and the donor positive are under increased risk for the development of cmv disease (9 – 12). most of the cmv-dna positive recipients in this study come from a recipient–donor pair in which both were cmv igg positive, 26 out of 30 in total, which is 86.7% of all positive recipients. the other 4 cmv-dna positive participants come from a recipient–donor pair in which the recipient was cmv-dna negative and the donor positive. considering the fact that recipient–donor pairs in which both were cmv igg positive far outnumbered participants with other serological combinations, it is necessary to mention the share of cmv-dna positive participants in each group. there were 84 participants belonging to a pair in which both the recipient and the donor were cmv igg positive, and out of them all, 26 were cmv-dna positive at some point after the tx, which constitutes 31% of such participants. seven participants belonged to a recipient– donor pair in which the recipient was cmv igg negative and the donor positive. among them, 4 participants became cmv-dna positive. the remaining 14 participants for whom the serostatus combination is known belonged to a recipient–donor pair in which the recipient was cmv igg positive and the donor was negative; none of them became cmv-dna positive. there were no recipient–donor pairs in which both were cmv igg negative. these findings coincide with previous studies from the pre-prophylaxis era, which studied the natural course of cmv infection in kidney transplant recipients and showed that 56% of kidney transplant recipients from a recipient–donor pair in which the recipient is cmv igg negative and the donor positive develop cmv disease after tx, as do 20% of those from a pair in which both the recipient and the donor are cmv igg positive (12). the participants of this study who received a kidney transplant from a cmv igg negative donor (all of them were cmv igg positive in this study), in addition to never becoming cmv-dna positive after the tx, also had better kidney transplant function compared to those who received their graft from a cmv igg positive donor. lack of viremia in such participants was accompanied by better kidney transplant function. there was no significant difference in the occurrence of cmv-dna positivity between the participants who took prophylaxis and those who did not, but cmv disease was more common in those who did not take it, with marginal significance (p = 0.066). however, with an insufficiently large sample of participants who did not take prophylaxis, the rarity of clinical seemedj 2021, vol 5, no. 1 cytomegalovirus in kidney transplantion 23 southeastern european medical journal, 2021; 5(1) manifestations of infections in relatively common viremia could still be interpreted as a consequence of administered prophylaxis and timely detection of viremia with regular testing for cmv-dna. out of 7 participants who had clinical symptoms, 2 developed cmv disease and 5 of them had cmv syndrome. one participant from the preprophylaxis era developed early invasive cmv disease and died as a result. one participant developed cmv disease, which manifested in elevated serum liver enzymes and esophagitis. of the participants who had cmv syndrome, 1 participant had elevated serum liver enzymes, 1 had leukopenia and 1 had weight loss. one participant had diarrhea, fever, and malaise, and 1 had diarrhea and loss of appetite. all of them, except for the deceased one, were successfully treated with antiviral medication. previous studies have shown that gastrointestinal symptoms were the most common manifestations of cmv infection and disease (3, 13), which coincides with our findings. participants were tested for cmv-dna once a year on average, but the frequency of such testing greatly varied from participant to participant, with some of them not being tested for years during certain periods. such findings, together with the relatively frequent cmv viremia in the observed population, and the significance which cmv infection has for graft function and overall survival of kidney transplant recipients, lead us to conclude that more work is needed to develop a standardized protocol for prevention of cmv infection in kidney transplant recipients. the limitations of this study, in the sense of evidence-based medicine, were the epidemiological character (design) and the absence of comparable data in a scenario without prophylaxis, since diagnostics, screening and follow-up of cmv were rare and deficient in the pre-prophylaxis era. other than that, there remains an important aspect of this problem, which could complement this study in the future. it is the immunosuppressive protocol that was part of the participants’ treatment, its dynamics over time, with respect to both the year of tx and the protocols in force during that time, as well as complications and comorbidities other than cmv. likewise, the promptness of the valganciclovir dose adjustment in relation to kidney function dynamics should be studied, although it appears adequately adjusted at the two studied points of time – at the beginning and at the end of administration of prophylaxis. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. bate s, dollard s, cannon m. cytomegalovirus seroprevalence in the united states: the national health and nutrition examination surveys, 1988 – 2004. clin infect dis. 2010;50:1439-47. https://pubmed.ncbi.nlm.nih.gov/20426575/ 2. cannon m, schmid d, hyde t. review of cytomegalovirus seroprevalence and demographic characteristics associated with infection. rev med virol. 2010; 20:202-13. 3. arthurs s, eid a, pedersen r, kremers w, cosio f, patel r, razonable rr. delayed-onset primary cytomegalovirus disease and the risk of allograft failure and mortality after kidney transplantation. clin infect dis. 2008; 46:840-6. 4. reischig t, jindra p, hes o, svecová m, klaboch j, treska v. valacyclovir prophylaxis versus preemptive valganciclovir therapy to prevent cytomegalovirus disease after renal transplantation. am j transplant. 2008;8:69-77. 5. hodson e, jones c, webster a, strippoli g, barclay p, kable k, vimalachandra d, craig jc. antiviral medications to prevent cytomegalovirus disease and early death in recipients of solidorgan transplants: a systematic review of randomised controlled trials. lancet. 2005; 365:2105-15. seemedj 2021, vol 5, no. 1 cytomegalovirus in kidney transplantion 24 southeastern european medical journal, 2021; 5(1) 6. strippoli g, hodson e, jones c, craig j. pre-emptive treatment for cytomegalovirus viremia to prevent cytomegalovirus disease in solid organ transplant recipients. transplantation. 2006; 81:139-45. 7. kalil a, levitsky j, lyden e, stoner j, freifeld a. meta-analysis: the efficacy of strategies to prevent organ disease by cytomegalovirus in solid organ transplant recipients. ann intern med. 2005; 143:870-80. 8. pilmore h, pussel b, goodman d. khacari guideline: cytomegalovirus disease and kidney transplantation. nephrology. 2011; 16:683-7. 9. abbott kc, hypolite io, viola r, poropatich rk, hshieh p, cruess d, hawkes ca, agodoa ly. hospitalizations for cytomegalovirus disease after renal transplantation in the united states. ann epidemiol. 2002; 12:402-9. 10. santos c, brennan d, fraser v, olsen m. delayed-onset cytomegalovirus disease coded during hospital readmission after kidney transplantation. transplantation. 2014; 98:187-94. 11. khoury j, storch g, bohl d, schuessler r, torrence s, lockwood m, gaudreault-keener m, koch mj, miller bw, hardinger kl, schnitzler ml, brennan dc. prophylactic versus preemptive oral valganciclovir for the management of cytomegalovirus infection in adult renal transplant recipients. am j transplant. 2006; 6:2134-43. 12. hartmann a, sagedal s, hjelmesaeth j. the natural course of cytomegalovirus infection and disease in renal transplant recipients. transplantation. 2006; 82:s15-7. 13. eid a, arthurs s, deziel p, wilhelm m, razonable r. clinical predictors of relapse after treatment of primary gastrointestinal cytomegalovirus disease in solid organ transplant recipients. am j transplant. 2009; 10:157-61. 14. kotton cn, kumar d, caliendo am, huprikar s, chou s, danziger-isakov l, humar a; the transplantation society international cmv consensus group. the third international consensus guidelines on the management of cytomegalovirus in solid-organ transplantation. transplantation. 2018 jun;102(6):900-931. 1 author contribution. acquisition of data: šisl d, zibar l. administrative, technical or logistic support: šisl d, zibar l. analysis and interpretation of data: šisl d, zibar l. conception and design: šisl d, zibar l. critical revision of the article for important intellectual content: šisl d, zibar l. drafting of the article: šisl d, zibar l. final approval of the article: šisl d, zibar l. guarantor of the study: šisl d, zibar l. provision of study materials or patients: šisl d, zibar l. statistical expertise (statistical analysis of data): šisl d, zibar l. seemedj 2021, vol 5, no. 1 life habits in dementia prevention 135 southeastern european medical journal, 2021; 5(1) original article role of life habits as a construct in dementia prevention1 ivana stanić 1*; silvija hinek 2; josip ivanda 2 1 university north, republic of croatia; osijek-baranja county 2 nursing home đakovo, republic of croatia # co-editor of the manuscript: s. lecturer mirna sabljar, phd; the academy of arts and culture in osijek, university of osijek, croatia *corresponding author: ivana stanić, ivana.stanic@obz.hr received: sep 17, 2020; revised version accepted: feb 10, 2021; published: apr 28, 2021 keywords: aging, dementia, healthy lifestyle abstract introduction: for over three decades, europe has been viewed as a continent of the elderly. however, despite the fact that the republic of croatia is a part of europe, it is only nowadays that we have come to face this problem in the form of a large – and rapidly growing – elderly population. since age is the main risk factor for the onset of dementia, a further increase in the number of patients is expected. consequently, the need for new insights in the field of constructs that contribute to the prevention of dementia is implied in the context of reducing the number of patients. this research aimed at gaining insight into life habits of individuals using homes for the elderly and infirm, which habits have been shown by previous research as contributory to dementia prevention or mitigation. materials and methods: this research was conducted using an appropriate sample of 443 users of decentralized homes for the elderly and infirm in osijek-baranja county. results: the obtained research data was divided into four categories: physical activity, cognitive activity, consumption of tobacco products, and alcohol and diet. the results showed that physical and cognitive activities are the least represented in everyday lives of users of homes for the elderly and infirm. conclusion: based on research results, the importance and role of physical activity and maintenance of cognitive skill will be emphasised with an aim of achieving better quality aging, especially in terms of dementia prevention in the elderly. (stanić i; hinek s; ivanda j. role of life habits as a construct in dementia prevention. seemedj 2021; 5(1); 135-144) seemedj 2021, vol 5, no. 1 life habits in dementia prevention 136 southeastern european medical journal, 2021; 5(1) introduction dementia describes a group of complex disorders that change and interfere with normal brain activities and result in disorders of various cognitive functions. one of the most common degenerative nervous system diseases from the dementia group is alzheimer’s disease. the disease is characterized by a progressive course, and in individuals older than 65 it is the most frequent cause of dementia (2). care for the elderly suffering from dementia, given the specific behavioural and psychological symptoms and physical illnesses, requires specific medical interventions and imposes a heavy burden of care. because of that, it is important to choose appropriate medical institutions that are able to cope with various care needs of individuals suffering from dementia (3). the incidence of alzheimer’s dementia is increasingly gaining the scale of a global health crisis and represents a substantial economic burden on society as a whole (4). since there are no specialized institutions for taking care of individuals suffering from various forms of dementia in the republic of croatia, it is necessary to focus on dementia prevention in order to reduce the number of individuals affected. a significant number of attempts in terms of therapeutic treatment have not been satisfactory, possibly because the treatment starts too late and at advanced stages of the disease, with highly developed pathology. research is focused on treatment, while prevention is neglected (5). therefore, an alternative strategy in the fight against dementia can be the prevention of various changeable factors, which also includes adequate control of comorbidities and lifestyle changes with avoidance of harmful habits (6). comprehensive clinical and epidemiological evidence points towards a close connection between dementia and heart disease, and due to their increased incidence and coexistence, they demand more attention because they represent a threat to public health (7). alzheimer’s disease is linked to smoking tobacco products since the risk of dementia is increased in the population of active smokers (8). type 2 diabetes, repeated head traumas, obesity, high blood pressure and social interactions are also changeable risk factors for dementia development. with early detection and measures for prevention of the aforementioned factors, it could potentially be possible to effectively prevent the development of alzheimer’s disease (2). adequate diet in combination with physical activity also represents a therapeutic potential in terms of prevention and delaying the onset of symptoms (4). research has confirmed that there are protective factors that contribute to dementia prevention or reduce its severity, for example, a higher level of education, intellectual activity, diet rich in unsaturated fatty acids, physical activity and moderate alcohol consumption (9). the theoretical and practical findings referred to above are the source from which the purpose of this research emerged. this research aims at getting an insight into life habits of users of homes for the elderly and infirm for which previous research has shown to contribute to dementia prevention or mitigation. three hypotheses have been set: a) the elderly do not recognize the importance of physical activity as one of the factors contributing to dementia prevention or mitigation; b) the elderly do not recognize the importance of maintaining cognitive activities as one of the factors contributing to dementia prevention or mitigation and c) the elderly do not recognize the importance of avoiding alcohol and tobacco products consumption as factors contributing to dementia prevention or mitigation. materials and methods the primary purpose and aim of this research is to determine which physical activities, cognitive activities and substances directly affect the life of the elderly, but at the same time contribute to development of dementia. for achieving the aforementioned goal, it was necessary to collect data and carry out the primary research process. the data was collected via an anonymous questionnaire designed by the authors. this research was conducted on an appropriate sample of 443 users of decentralized homes for seemedj 2021, vol 5, no. 1 life habits in dementia prevention 137 southeastern european medical journal, 2021; 5(1) the elderly and infirm in osijek-baranja county. the questionnaire created by the authors for the purposes of this research was used by interviewing users in person, by interviewers. statistical analysis analyses of the collected data were performed using the statistical program ibm spss statistics 23 for social sciences. in order to examine the correlation between the variables included in this study, pearson correlation coefficients were calculated. the respective correlation coefficients, together with their significance, are shown in the results. table 1. socio-demographic characteristics of research participants n (%) participants’ gender female 317 (71.6%) male 126 (28.4%) participants’ age under 50 3 (0.7%) 51-60 8 (1.8%) 61-70 29 (6.5%) 71-80 169 (38.2%) 81-90 207 (46.8%) 91-100 27 (6%) qualifications no elementary education 86 (19.4%) elementary education 151 (34.1%) secondary education 151 (34.1%) specialist training 40 (9%) higher education 14 (3.2%) doctorate 1 (0.2%) time spent in a home or homes for the elderly and infirm less than 1 year 52 (11.7%) 1-5 years 211 (47.6%) 6-10 years 120 (27.1%) over 10 years 60 (13.5%) source: authors seemedj 2021, vol 5, no. 1 life habits in dementia prevention 138 southeastern european medical journal, 2021; 5(1) results socio-demographic data this research included 443 participants who are users of homes for the elderly and infirm in osijek-baranja county. in terms of gender, 317 (71.6%) were women and 126 (28.4%) were men. regarding age, 3 participants (0.7%) were under 50 years of age, 8 (1.8%) were between 51 and 60 years, 29 (6.5%) were between 61 and 70 years, 169 (38.2%) were between 71 and 80 years, 207 (46.8%) were between 81 and 90 and 27 (6%) were between 91 and 100 years of age. regarding qualifications, 86 (19.4%) of the participants have not finished elementary school, 151 (34.1%) have completed their elementary education and the same number (151, i.e., 34.1%) have completed their secondary education, 40 (9%) have finished specialist training, 14 (3.2%) have higher education degrees and only one participant (0.2%) has a doctorate. since all participants were users of homes for the elderly and infirm, they differed by time spent in homes; 52 (11.7%) participants had lived in a home less than one year, 211 (47.6%) participants had lived in a home between 1 and 5 years, 120 (27.1%) participants had lived in a home between 6 and 10 years, and 60 (13.5%) had lived in a home for over 10 years (table 1). physical activity table 2 presents data regarding physical activity of participants. when asked: “how often do you exercise?”, as many as 168 participants (37.9 %) answered “never”, 160 participants (36.1%) answered “occasionally”, 76 participants (17.2%) answered “regularly”, and only 39 participants (8.8%) answered “often”. when asked: “how often do you take walks in nature?”, 74 participants (16.7%) answered “never”, 168 participants (37.9%) reported that they do it “occasionally”, 117 participants (26.4%) answered “regularly”, and 84 participants (19%) answered that they often take walks in nature. furthermore, as many as 148 (33.4%) of participants never use stairs instead of an elevator, 112 participants (25.3%) occasionally use stairs, 83 participants (18.7%) use stairs regularly, and 100 participants (22.6%) frequently use stairs instead of an elevator... table 2. descriptive data for physical activity parameters n (%) how often do you exercise? never 168 (37.9%) occasionally 160 (36.1%) regularly 76 (17.2%) often 39 (8.8%) how often do you take walks in nature? never 74 (16.7%) occasionally 168 (37.9%) regularly 117 (26.4%) often 84 (19%) how often do you use stairs instead of an elevator? never 148 (33.4%) occasionally 112 (25.3%) regularly 83 (18.7%) often 100 (22.6%) source: authors seemedj 2021, vol 5, no. 1 life habits in dementia prevention 139 southeastern european medical journal, 2021; 5(1) cognitive activity table 3 presents data regarding cognitive activity of participants. namely, when asked: “how often do you solve crosswords, do puzzles or play memory games?”, as many as 258 participants (58.2%) answered “never”, 60 participants (13.5%) answered “occasionally”, 62 participants (14%) answered “regularly”, and 63 participants (14.2%) answered “often”. furthermore, when asked: “how often do you play chess?”, 318 participants (71.8%) answered “never”, 48 participants (10.8%) answered “occasionally”, 34 participants (7.7%) answered “regularly” and 43 (9.7%) answered “often”. when asked: “how often do your read books, magazines, newspapers?”, 156 participants (35.2%) answered “never”, 117 participants (26.4%) answered “occasionally”, 82 participants (18.5%) answered “regularly”, and 88 (19.9%) answered that they often read books, magazines and newspapers. table 3. descriptive data for cognitive activity parameters n (%) how often do you solve crosswords, do puzzles or play memory games? never 258 (58.2%) occasionally 60 (13.5%) regularly 62 (14%) often 63 (14.2%) how often do you play chess? never 318 (71.8%) occasionally 48 (10.8%) regularly 34 (7.7%) often 43 (9.7%) how often do your read books, magazines, newspapers? never 156 (35.2%) occasionally 117 (26.4%) regularly 82 (18.5%) often 88 (19.9%) source: authors consumption of alcohol and tobacco products table 4 presents data that show that 312 participants (70.4%) never consume tobacco products, 30 participants (6.8%) consume them occasionally, 44 participants (9.9%) consume them regularly, and 57 participants (12.9%) consume them often. furthermore, from table 4 we see that 295 participants (66.6%) never consume alcohol, 76 participants (17.2%) consume it occasionally, 26 participants (5.9%) consume it regularly, and 46 participants (10.4%) consume it often. seemedj 2021, vol 5, no. 1 life habits in dementia prevention 140 southeastern european medical journal, 2021; 5(1) table 4. descriptive data for consumption of tobacco products and alcohol parameters n (%) how often do you consume tobacco products? never 312 (70.4%) occasionally 30 (6.8%) regularly 44 (9.9%) often 57 (12.9%) how often do you consume alcohol? never 295 (66.6%) occasionally 76 (17.2%) regularly 26 (5.9%) often 46 (10.4%) source: authors diet table 5 shows data regarding the participants’ diet. salads, fresh fruit and vegetables are never consumed by 13 participants (2.9%), 90 participants (20.3%) eat them occasionally, 260 participants (58.7%) eat them regularly, and 78 participants (17.6%) often eat salads and fresh fruit and vegetables. two participants did not provide an answer to this question. regarding fish, olive oil, nuts and eggs, 43 participants (9.7%) never consume them, 161 participants (36.3%) consume them occasionally, 192 participants (43.3%) consume them regularly, and 47 participants (10.6%) consume them often. when asked: “how often do you consume bakery products, sweets and salty foods?”, 54 participants (12.2%) answered “never”, 172 participants (38.8%) answered “occasionally”, 145 participants (32.7%) answered “regularly”, and 72 participants (16.3%) answered they consume these products often.. table 5. descriptive data for dietary parameters n (%) how often do you consume tobacco products? never 312 (70.4%) occasionally 30 (6.8%) regularly 44 (9.9%) often 57 (12.9%) how often do you consume fish, olive oil, nuts, eggs? never 43 (9.7%) occasionally 161 (36.3%) regularly 192 (43.3%) often 47 (10.6%) how often do you consume bakery products, sweets and salty foods? never 54 (12.2%) occasionally 172 (38.8%) regularly 145 (32.7%) often 72 (16.3%) source: authors seemedj 2021, vol 5, no. 1 life habits in dementia prevention 141 southeastern european medical journal, 2021; 5(1) correlation between variables table 6 shows significant negative and insignificant correlations between the variables “how often do you walk in nature?” and “time spent in a home or homes for the elderly and infirm” (r = -0.11; p = 0.027) and variables “how often do you consume tobacco products?” and “time spent in a home or homes for the elderly and infirm” (r = -0.10; p = 0.029). other parameters of physical and cognitive activity did not prove to be significantly correlated to the length of stay in a home or homes for the elderly and infirm, nor did alcohol consumption and eating habits. table 6. review of correlations of variables time spent at a home or homes for the elderly and infirm how often do you exercise? -0.04 how often do you take walks in nature? -0.11* how often do you solve crossword puzzles, do puzzles or play memory games? -0.04 how often do you play chess? -0.09 how often do you read books, magazines, newspapers? -0.06 how often do you consume tobacco products? -0.10* how often do you consume alcohol? -0.08 how often do you eat salads, fresh vegetables and fruit? -0.01 how often do you consume fish, olive oil, nuts, eggs? -0.06 how often do you consume bakery products, sweets and salty foods? 0.05 how often do you climb the stairs instead of using an elevator? -0.09 how often do you use medications? 0.06 note: *p<0.05; **p<0.01 (significant correlations are written in bold) source: authors discussion the obtained research data was divided into four categories: physical activity, cognitive activity, consumption of tobacco products, and alcohol and diet. the results showed that physical and cognitive activities are the least represented in everyday lives of users of homes for the elderly and infirm, i.e., that the elderly do not recognize the significance of physical and cognitive activities as important factors that contribute to dementia prevention or mitigation, which confirms hypothesis a) and hypothesis b). positive aspects are that the majority of participants do not consume tobacco products or alcohol, which rejects hypothesis 3, considering that the elderly do recognize the importance of avoiding the consumption of tobacco products and alcohol as important factors that contribute to dementia prevention or mitigation. furthermore, diet differs when it comes to individual items so the participants in most cases consume fresh fruit and vegetables, but they rarely consume fish, olive oil, nuts and eggs. furthermore, in showing the correlations between the variables, the results showed that there is a significant, but only slightly significant, correlation between the variables related to the frequency of taking walks in nature and consuming tobacco products with the time spent in a home. namely, the longer the time spent in a home or homes for the elderly and infirm, the less frequently the users take walks in nature and consume tobacco products. previous research has shown that nonpharmacological interventions could have a significant role in prevention, but also in the progression of dementia (10). research was mostly focused on the treatment of dementia, while prevention was neglected (5), but lately, seemedj 2021, vol 5, no. 1 life habits in dementia prevention 142 southeastern european medical journal, 2021; 5(1) due to the increasing number of affected individuals, dementia is becoming a public health problem and a larger number of researchers focus on detecting risk and protective factors in dementia prevention. according to mimica, we cannot protect ourselves completely against dementia, but if simple recommendations are adopted, the risk of developing dementia can be reduced by one third (11). in a research conducted by farina et al. based on six observed studies, it was concluded that physical activity has a positive effect on increase in cognitive functions in the case of dementia (10). du et al. conducted a research on 869 participants suffering from alzheimer’s disease. their results also showed positive effects of physical activity on cognitive functions of patients (12). the fact that an active lifestyle is one of the predictors of dementia prevention or mitigation was also confirmed by a research conducted by rolland et al. (13). they claimed that in the future, prevention of alzheimer’s disease could be based on precisely determined rules regarding management of life habits that include physical activity, cognitive activity and diet (13). focusing on the principle that physical activity contributes to dementia prevention, sondell et al. conducted a research in 16 homes for the elderly and infirm in order to observe how exercise programs for the users should be created. their results showed that group, monitored and individual programs of functional training are the most effective for dementia prevention and that it is very important to motivate users of homes for the elderly and infirm to participate in such programs (14). furthermore, rege et al. conducted a study that examined 164 epidemiological, longitudinal, cross-sectional, intervention and randomized controlled studies. results of this overview confirmed therapeutic potential of physical activity in combination with diet as an important protective factor in dementia prevention (4). according to kornhuber, a healthy lifestyle with daily activities performed outdoors, mediterranean diet and reduced consumption of alcohol significantly reduce the risk of dementia, in which context the results showed that cognitively stimulating activities protect even more than physical activities (5). the fact that cognitive engagement, regular physical activity, mediterranean diet and the consumption of omega-3 fatty acids are important protective factors in dementia prevention was confirmed by a study conducted by barak and aizenberg (15). furthermore, most users come to institutional care when they become dependent on the help and care of others due to their health condition, which reduces their mobility, but research conducted by močnik et al. (17) shows that even mobile users are not aware of the importance of physical activity and its effects on health in old age and that 71% of respondents do not participate in any physical activity in homes for the elderly and infirm (17). this is why the emphasis should be on motivating and informing users about healthy lifestyles and encouraging lifestyle changes. in addition, literature shows that smoking is significantly linked to an increased risk of alzheimer’s disease. namely, smoking-related cerebral oxidative stress is a potential mechanism that promotes the pathophysiology of alzheimer’s disease and increases the risk of developing this disease (16). finally, all conducted studies indicate that dementia prevention is possible. namely, physical and cognitive activity, mediterranean diet and reduced consumption of alcohol and tobacco are key protective factors in the fight against this disease. the results of this research raise the question about the reason why the elderly have life habits shown in the results. why is it the case that the majority of the elderly do not recognize the importance of physical and cognitive activity as protective factors in dementia prevention? since all the participants live in homes for the elderly and infirm, the results of this research place a challenge in front of experts employed at those institutions, but also in front of other stakeholders, such as political and public authorities that can contribute to the creation of strategies and programs for dementia prevention. the elderly need to be educated and provided with information on the importance of changing life habits with an aim of dementia prevention. it is also important to note seemedj 2021, vol 5, no. 1 life habits in dementia prevention 143 southeastern european medical journal, 2021; 5(1) that, given that the participants were users of homes for the elderly and infirm, where alcohol and tobacco consumption is prohibited, it is possible that this policy influenced the research results and the rejection of hypothesis 3. namely, the results show that a significant majority of participants never consume alcohol and tobacco products and that the frequency of consumption of tobacco products decreases as time spent at a home increases, which could be the result of obeying house rules of a home they reside in. furthermore, regarding diet in homes for the elderly and infirm, users receive already prepared meals and cannot choose their menus. because of that, diet standards in institutions should be harmonized based on the nutrients needed for providing the highest possible level of protective factors for dementia prevention. cooperation between public authorities and homes for the elderly and infirm with an aim of dementia prevention will give results over a certain period, and we could use a longitudinal research in order to examine the extent to which life habits of the users have changed. upon consolidation of author’s research and the mentioned previous research, it can be concluded that physical activities, cognitive activities and substances that directly affect the lives of the elderly are imperative in terms of life habits that as a construct have a role in dementia prevention. on the other hand, we can say that the research has brought to light significant segments of risky behaviour of users that are of crucial importance, and as such can contribute to dementia process. staff communication in the process of everyday interaction with users must be given primacy, through training with a view of improving the users’ quality of life. communication also has a role of transferring information, instructing and activating users; consequently, emphasis has to be on educated staff that plays a daily role in the lives of users of homes for the elderly and infirm. acknowledging the fact that the elderly have a higher risk of developing some type of dementia, it has been confirmed that dementia prevention is possible and necessary in preserving the quality of life of the elderly. obtained research results can give a special contribution to future research by the authors, at which occasion initial and new discoveries will be compared, after allowing some time for guidelines to be given to the elderly through direct communication with experts. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. world health organization. dementia. http://www.euro.who.int/en/healthtopics/noncommunicable-diseases/mentalhealth/areas-of-work/dementia (12.01.2020). 2. borovečki f. early diagnosis of alzheimer’s disease. medicus 2019; 28 (suppl 1): 23-26. 3. hattori h. role of geriatric hospitals for dementia care in the community. psychogeriatrics. 2012; 12 (suppl 2):124-126. 4. rege sd, geetha t, broderick tl, babu jr. can diet and physical activity limit alzheimer’s disease risk? curr alzheimer res. 2017; 14 (suppl 1):76-93. 5. kornhuber hh. prevention of dementia (including alzheimer's disease). gesundheitswesen. 2004; 66 (suppl 5):346-351. 6. edwards iiiga, gamez n, escobedo gjr, calderon o, moreno-gonzalez i. modifiable risk factors for alzheimer's disease. front aging neuroscience. 2019; 11 (suppl 146):1-18. 7. yang m, li c, zhang y, ren j. interrelationship between alzheimer's disease and cardiac dysfunction: the brain-heart continuum? acta biochim biophys sin 2020; 52( suppl 1): 1-8. 8. reitz c, den heijer t, van duijn c, hofman a., breteler mmb. relation between smoking and risk of dementia and alzheimer disease. neurology. 2007; 69 (suppl 10):998-1005. seemedj 2021, vol 5, no. 1 life habits in dementia prevention 144 southeastern european medical journal, 2021; 5(1) 9. mayeux r, stern y. epidemiology of alzheimer disease. cold spring harbor perspectives in medicine. 2012; 2 (suppl 8):10. 10. farina n, rusted j, tabet n. the effect of exercise interventions on cognitive outcome in alzheimer's disease: a systematic review. psychogeriatrics. 2014; 26 (suppl 1):9-18. 11. mimica n. is the prevention of dementia possible? in: abstracts of the 2th alzheimer’s educational conference edukal, zagreb, 2016. p. 12. croatian alzheimer’s association, zagreb, croatia. 12. du z, li y, li j, zhou c, li f, yang x. physical activity can improve cognition in patients with alzheimer's disease: a systematic review and meta-analysis of randomized controlled trials. clin interv aging 2018; 4 (suppl 13):1593-1603. 13. rolland y, abellan van kan g, vellas b. physical activity and alzheimer's disease: from prevention to therapeutic perspectives. j am med dir assoc. 2008; 9 (suppl 6):390-405. 14. sondell a, rosendahl e, gustafson y, lindelof n, littbrand h. the applicability of a high-intensity functional exercise program among older people with dementia living in nursing homes. j geriatr phys ther. 2019; 42 (suppl 4):16-24. 15. durazzo tc, mattsson n, weiner mw. smoking and increased alzheimer’s disease risk: a review of potential mechanisms. alzheimers dementia 2014; 10 (suppl 3):122-145. 16. barak y, aizenberg d. is dementia preventable? focus on alzheimer's disease. exp rev neuroth 2010; 10 (suppl 11):1689-1698. 17. močnik a, neuberg m, canjuga i. physical activity of elderly people accommodated in residential institutions. technical j 2015; 9 (suppl 1):112-119.. author contribution. author contribution. acquisition of data: stanić i; hinek s; ivanda j administrative, technical or logistic support: stanić i; hinek s; ivanda j analysis and interpretation of data: stanić i; hinek s; ivanda j critical revision of the article for important intellectual content: stanić i; hinek s; ivanda j drafting of the article: stanić i; hinek s; ivanda j final approval of the article: stanić i; hinek s; ivanda j guarantor of the study: stanić i; hinek s; ivanda j provision of study materials or patients: stanić i; hinek s; ivanda j statistical expertise (statistical analysis of data): stanić i; hinek s; ivanda j seemedj 2020, vol 4, no. 2 tff3 expression after chemotherapy 20 southeastern european medical journal, 2020; 4(2) original article neoadjuvant chemotherapy affects tff3 peptide expression in luminal b subtype of breast cancer – a pilot study 1 nikola bijelić *1, martina abramović 2, jasmina rajc 3, edi rođak 1, zlatko marušić 2, maja tolušić levak 1, 4, biljana pauzar 1, 5, tatjana belovari 1 1 department of histology and embryology, faculty of medicine, university of osijek, croatia 2 department of pathology and cytology, university hospital centre zagreb, croatia 3 department of pathology and cytology, university hospital centre osijek, croatia 4 department of dermatology and venerology, university hospital centre osijek, croatia 5 department of clinical cytology, university hospital centre osijek, croatia *corresponding author: nikola bijelić, nbijelic@mefos.hr received: mar 12, 2020; revised version accepted: oct 23, 2020; published: nov 12, 2020 keywords: breast cancer; immunohistochemistry; neoadjuvant therapy; tff3 peptide abstract aim: trefoil factor family 3 (tff3) peptide is normally expressed by epithelial cells in breast ducts, but it is also associated with different pathological conditions, including breast cancer. it is considered a marker of poor prognosis and associated with increased resistance to chemotherapy. data on the effect of chemotherapy on tff3 peptide expression are scarce. the aim of this pilot study was to assess suitability of research on this topic for large-scale studies. methods: formalin-fixed, paraffin-embedded samples of core biopsies and of surgically removed tumors from patients with luminal b subtype of breast cancer were used for immunohistochemical analysis. changes in tff3 peptide and ki-67 expression and microvessel density (mvd) values before and after chemotherapy were analyzed, as well as the association between tff3 peptide expression and ki-67 expression and mvd values. results: significant reduction in tff3 and ki 67 expression was observed after chemotherapy, while mvd values did not differ significantly before and after chemotherapy. the association of tff3 peptide expression and ki-67 expression and tff3 peptide expression and mvd values was not significant before or after chemotherapy. conclusion: the data obtained in this pilot study suggest that a large-scale study is justified, and it other breast cancer subtypes should be included. (bijelić n, abramović m, rajc j, rođak e, marušić z, tolušić levak m, pauzar b, belovari t. neoadjuvant chemotherapy affects tff3 peptide expression in luminal b subtype of breast cancer – a pilot study. seemedj 2020; 4(2); 20-27) seemedj 2020, vol 4, no. 2 tff3 expression after chemotherapy 21 southeastern european medical journal, 2020; 4(2) introduction early detection of breast cancer enables better treatment. also, adequate methods for monitoring response to treatment help in planning further treatment. modern breast cancer diagnostics include core biopsy with immunohistochemical characterization of tumor tissue. according to st. galen consensus, breast cancer is classified into following surrogate subtypes: luminal a, her2-positive luminal b, her2-negative luminal b, her2-enriched and triple negative breast cancer. such classification helps with prognosis and choice of therapy and is based on expression of estrogen receptor, progesterone receptor, her2 and ki 67 in the tumor [1]. still, there is a need for further characterization of oncogenes and molecular markers connected to the onset, progression and relapse of breast cancer [2]. in modern breast cancer therapy, neoadjuvant chemotherapy is considered a standard approach in an increasing number of clinical settings (e. g. in patients with tumor subtypes that are highly sensitive to chemotherapy and in patients that have tumors > 2 cm and/or metastases in axillar lymph nodes), also allowing for cancer down-staging and helping with breast conservation [3,4]. it also alters expression of different markers in cancer cells, such as hormone receptors, her2, claudin-1 and claudin3 [5,6]. trefoil factor family (tff) is a group of three small secretory peptides – tff1, tff2 and tff3 peptide, mostly secreted by epithelial cells of various mucosal sites. their major role is mucosal protection, and they are known to affect cell migration, apoptosis, angiogenesis and immune response [7]. of the three peptides, tff3 peptide is the most widespread in the human organism, and is normally expressed by epithelial cells in breast ducts [8,9]. however, presence of tff peptides is also associated with different pathological conditions, including breast cancer. tff3 peptide is expressed in different subtypes of breast cancer, most predominantly in hormone receptor-positive subtypes and is considered a marker of poor prognosis because it promotes metastatic seeding and neoangiogenesis [10,11]. our previous research showed that tff3 peptide expression was the highest in luminal b subtype, moderately differentiated cancers (grade ii) and in tumors with moderate expression of ki 67, implying that tff3 peptide might be considered a possible marker for determining tumor status, which might influence the choice of therapy [12]. presence of tff3 peptide in breast cancer cells is associated with increased resistance to chemotherapy, while cancers with complete response to chemotherapy have lower or no tff3 peptide expression [13]. despite the growing interest in tff3 peptide research in the context of breast cancer diagnostics and therapy, there are no data on the effect of chemotherapy on tff3 peptide expression in breast cancer tissue. therefore, we launched a pilot study in order to assess the suitability of a large-scale study. material and methods study design and material this was a cross-sectional pilot study performed on tissue samples archived at the department of pathology and cytology, university hospital centre zagreb. tissue samples of luminal b subtype of breast cancer were used in the study. they were taken during routine diagnostic and therapy procedures from women whose treatment included neoadjuvant chemotherapy. there were 22 tumor samples overall; 10 were her2-negative, and 12 were her2-positive. also, samples from 5 normal breast tissue controls were included for comparison of tff3 peptide expression. for each tumor, a core biopsy sample (pre-chemotherapy) and a sample of surgically removed tumor (post-chemotherapy) were used. chemotherapy protocols of each patient are available in the supplemental table. changes in tff3 peptide and ki-67 expression and in microvessel density (mvd) values before and after chemotherapy were analyzed, as well as the association between tff3 peptide expression and ki-67 expression and mvd values. seemedj 2020, vol 4, no. 2 tff3 expression after chemotherapy 22 southeastern european medical journal, 2020; 4(2) immunohistochemical staining for each patient, archived immunostained slides (hormone receptor positivity, ki-67 status and her2 status) were revised in order to confirm the cancer subtype. paraffin blocks of samples that met the criteria to be included in the study (luminal b subtype) were cut on a microtome (leica, sm2000r, leica biosystems, nussloch, germany) into 3-5-µm-thick slides and mounted onto adhesive slides suitable for immunohistochemistry. slides were stained for tff3 peptide and cd34 endothelial marker, and ki-67 staining scores (share of immunopositive nuclei) were obtained from the aforementioned archived slides. antigen retrieval was performed using the dako pt link device (dako, agilent technologies, santa clara, usa) with the envision flex target retrieval solution, low ph for tff3 peptide and ki-67 staining, and the dako envision flex target retrieval solution, high ph for cd34 staining. after that, the slides were stained in the dako techmate device using standard protocols as per the manufacturer’s instructions. purified monoclonal mouse antihuman primary antibody to tff3 peptide was used at 1:1000 dilution (sigma-aldrich, st. louis, usa), for ki-67 monoclonal mouse, anti-human antibody was used at 1:75 dilution (dako), and for cd34 monoclonal mouse, anti-human antibody was used at 1:50 dilution (dako). for immunostaining visualization, the dako envision flex kit was used. all slides were counterstained with hematoxylin, dehydrated and coverslipped using the entellan covering medium (merck, darmstadt, germany). for tff3 peptide staining, appendix tissue was used as positive control, while placenta tissue and the appendix tissue with primary antibody omitted were used as negative control. the appendix tissue was used as positive and negative control for cd34 (primary antibody omitted on negative controls). tff3 peptide tissue expression level was assessed using a modified quick score method that incorporates the share of immunopositive cells as well as staining intensity and reports the staining on a scale of 0 to 7 [12,14]. cd34 staining was used to obtain mvd (number of small blood vessels per surface unit under a 10 × objective) [15]. digital photographs were taken using the axiocam 305 color camera mounted on a zeiss axio imager a2 light microscope and zen 2.3 lite (blue edition) software (carl zeiss microscopy gmbh, jena, germany). the investigations were carried out following the rules of institutional and national ethical standards and the rules of helsinki declaration of 1975, revised in 2013. the study has been approved by the ethical committees of the university hospital centre zagreb (class: 8.118/256-2, no.: 02/21 a6) and the faculty of medicine osijek (no. 2158-61-07-19-10). statistical analysis patient age was expressed using mean values and standard deviation, while all other data were expressed using the median and interquartile range. for comparison of dependent samples, the non-parametric wilcoxon test was used, and the kendall's tau test was used for the analysis of the association of numerical variables. coefficient of determination was obtained by linear regression after correlation testing. for comparison of independent samples, the mannwhitney u test was used. data on ki 67 expression after chemotherapy were unavailable for two patients, and these were excluded from the analyses that included ki 67 expression. the statistical analysis was done using the medcalc software (18.11.6, medcalc software bvba, ostend, belgium) and a p-value less than 0.05 was considered statistically significant. results the mean age of cancer patients was 53.1 years (st. dev. 13.2) at the time of diagnosis; the mean age of subjects with no cancer was 55.6 years (st. dev. 9.07). tff3 peptide staining was detected both in breast cancer tissue and in normal breast tissue. the analysis of quick score values for tff3 peptide expression in patients with luminal b subtype of breast cancer showed a significant seemedj 2020, vol 4, no. 2 tff3 expression after chemotherapy 23 southeastern european medical journal, 2020; 4(2) reduction in tff3 peptide immunostaining after chemotherapy (table 1, figure 1). table 1. tff3 peptide expression, ki 67 expression and mvd values in luminal b breast cancer subtype before and after chemotherapy n before chemotherapy after chemotherapy p-value* tff3 quick score 22 6 (6 – 7) 5 (4 – 6) 0.005 ki-67 score (%) 20 31 (25 – 38.5) 11.5 (4.8 – 21) < 0.001 mvd value (/mm2) 22 79.9 (62.3 – 96.3) 71.3 (54.8 – 99.4) 0.59 * wilcoxon test figure 1. immunohistochemical staining of luminal b breast cancer tissue for tff3 peptide a) her2-positive cancer before chemotherapy (core biopsy), quick score = 7; b) the same tumor after chemotherapy (surgical excision), quick score = 3; c) her2-negative cancer before chemotherapy (core biopsy), quick score = 6; d) the same tumor after chemotherapy (surgical excision), quick score = 4. scale bar: 0.1 mm seemedj 2020, vol 4, no. 2 tff3 expression after chemotherapy 24 southeastern european medical journal, 2020; 4(2) compared to normal breast epithelium, quick score values did not differ significantly in breast cancer before chemotherapy, while after chemotherapy, the median quick score value was lower and close to significant values (p = 0.92 and 0.08, respectively; mann-whitney u test). ki 67 expression in patients with breast cancer was significantly lower after chemotherapy (table 1). microvessels were identified by cd34 staining. on several slides, tissue fibroblasts caused some false positivity, which was excluded from mvd calculation. mvd values did not differ significantly before and after chemotherapy (table 1). supplemental table 1. the table shows all cancer patients with their corresponding neoadjuvant therapy regimens and durations used in this study. patient code neoadjuvant therapy regimen (type and duration; notes) 2 her2ac-t 6 months 3 her2ac-t 6 months 4 her2ac-t 6 months 5 her2ac-t 6 months 6 her2ac-t 5 months 7 her2ac-t 7 months 9 her2ac-t 6 months 10 her2ac-t 6 months 11 her2ac-d (3 x ac + 1 x d) 2 months; discontinued due to intolerance 12 her2ac-d 2 months 1 her2+ acdd 2 months + php 2 her2+ acdd-thp* 6 months 3 her2+ ac-t 5 months**; trastuzumab 7 months 4 her2+ ac-t 6 months 6 her2+ ac-thp 6 months 8 her2+ ac-php 6 months 9 her2+ ac-php 5 months 10 her2+ ac-th 7 months 11 her2+ ac-php 6 months 12 her2+ ac-t + 4 ph 7 months 13 her2+ ac-t + ph duration unknown 14 her2+ ac-thp 6 months * trastuzumab taken for only 3 months ** regimen discontinued after the 4th paclitaxel dose abbreviations: ac-d – doxorubicin, cyclophosphamide, docetaxel; acdd – dose-dense ac regimen; ac-t – doxorubicin, cyclophosphamide, paclitaxel; ac-ph – doxorubicin, cyclophosphamide, pertuzumab, trastuzumab; ac-php: doxorubicin, cyclophosphamide, paclitaxel, trastuzumab, pertuzumab; ac-th – doxorubicin, cyclophosphamide, paclitaxel, trastuzumab; ac-thp – doxorubicin, cyclophosphamide, docetaxel, trastuzumab, pertuzumab seemedj 2020, vol 4, no. 2 tff3 expression after chemotherapy 25 southeastern european medical journal, 2020; 4(2) the association of tff3 peptide expression and ki 67 expression, as well as of tff3 peptide expression and mvd values, was not significant before or after chemotherapy, although the pvalue for the association of tff3 peptide expression and mvd values before chemotherapy was close to the significance level (table 2). table 2. association of tff3 peptide with ki-67 expression and mvd values in luminal b breast cancer subtype before and after chemotherapy tau 95% ci of tau pvalue* r2 before chemotherapy tff3 ki-67 0.04 -0.27 – 0.40 0.84 0.017 mvd 0.28 -0.19 – 0.58 0.07 0.090 after chemotherapy tff3 ki-67 -0.25 -0.52 – 0.16 0.11 0.031 mvd 0.25 -0.11 – 0.53 0.11 0.074 * kendall's tau test discussion this pilot study revealed a significant reduction in tff3 peptide and ki 67 expression after neoadjuvant chemotherapy, while no significant changes in mvd values were found. until now, no data have been available on the effect of chemotherapy on tff3 peptide expression in breast cancer cells or its association with ki-67 and mvd before and after chemotherapy. since it is known that overexpression of tff3 peptide affects proliferation of breast cancer cells, stimulates angiogenesis and contributes to chemotherapy resistance, information about the effect of chemotherapy on tff3 peptide expression should be helpful in understanding its role in breast cancer [11,13]. although expression of tff3 peptide increases the chances of local invasion and metastatic seeding, its role in breast cancer is still not fully understood [10]. some researchers argue that it should not be considered an oncogene, but a normal breast protein “abused” by cancer cells, and similar conclusions could be drawn from other diseases involving tff3 peptide expression [9,16]. due to its effect on disease progression and chemotherapy results, tff3 peptide is considered a potential therapeutic target in certain types of breast cancer, and current research looks promising [13,17,18]. nevertheless, since tff3 peptide has important physiological roles in the human organism, a careful risk/benefit assessment is needed. for example, tff3 peptide is important for maintenance of intestinal mucosa, which is often affected by chemotherapy, hence targeting tff3 peptide might worsen certain chemotherapy side-effects [19]. newly-formed small blood vessels can be an early predictor of metastatic potential, and cd34 is one of the most important endothelial markers used for small vessel detection [20]. mvd values did not change significantly in this research, and the correlation between mvd and tff3 quick score was weak. hence, although tff3 peptide stimulates angiogenesis, it seems that a reduction in tff3 expression after chemotherapy did not affect tumor vascularity. a reduction in ki 67 expression was expected, although it was interesting to see that there was no significant association between tff3 peptide and ki 67 expression before or after chemotherapy, probably due to different mechanisms regulating their expression. limitations to this pilot study were the analysis of only one subtype of breast cancer and sample size. nevertheless, based on the data obtained, we believe a large-scale study including other breast cancer subtypes is justified. since tff3 is a secretory protein found in different body liquids, there is also a possibility seemedj 2020, vol 4, no. 2 tff3 expression after chemotherapy 26 southeastern european medical journal, 2020; 4(2) of a quantitative evaluation of tff3 levels (e.g. in serum) by diagnostic methods. such sampling is less invasive than a core or surgical biopsy and could be used as a screening method in certain conditions, which should be further investigated in the future. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. goldhirsch a, winer ep, coates as, gelber rd, piccart-gebhart m, thürlimann b, senn h-j, panel members. personalizing the treatment of women with early breast cancer: highlights of the st gallen international expert consensus on the primary therapy of early breast cancer 2013. ann oncol 2013; 24: 2206–23. doi: 10.1093/annonc/mdt303 2. inoue k, fry ea. novel molecular markers for breast cancer. biomark cancer 2016; 8: 25–42. doi: 10.4137/bic.s38394 3. bartsch r, bergen e, galid a. current concepts and future directions in neoadjuvant chemotherapy of breast cancer. memo 2018; 11: 199–203. doi: 10.1007/s12254-018-0421-1 4. cardoso f, kyriakides s, ohno s, penaultllorca f, poortmans p, rubio it, zackrisson s, senkus e. early breast cancer: esmo clinical practice guidelines for diagnosis, treatment and follow-up. ann oncol 2019; 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11: 1528. doi: 10.3390/cancers11101528 19. beck pl, wong jf, li y, swaminathan s, xavier rj, devaney kl, podolsky dk. chemotherapyand radiotherapy-induced intestinal damage is regulated by intestinal trefoil factor. gastroenterology 2004; 126: 796– 808. 20. zaha dc. significance of immunohistochemistry in breast cancer. world j clin oncol 2014; 5: 382–92. doi: 10.5306/wjco.v5.i3.382 i i author contribution acquisition of data: bijelić n, abramović m, rajc j, marušić z administrative, technical or logistic support: bijelić n, abramović m, rajc j, rođak e, marušić z, tolušić levak m, pauzar b, belovari t analysis and interpretation of data: bijelić n, abramović m, rajc j, rođak e, marušić z, tolušić levak m, pauzar b, belovari t conception and design: bijelić n, abramović m, rajc j, marušić z, tolušić levak m, pauzar b critical revision of the article for important intellectual content: bijelić n, rajc j, rođak e, marušić z, tolušić levak m, pauzar b, belovari t drafting of the article: bijelić n, rođak e, tolušić levak m, belovari t final approval of the article: bijelić n, rajc j, rođak e, tolušić levak m, pauzar b, belovari t guarantor of the study: abramović m, rođak e, marušić z, tolušić levak m, pauzar b, belovari t provision of study materials or patients: bijelić n, rajc j,abramović m, marušić z, tolušić levak m statistical expertise: bijelić n, abramović m, rajc j seemedj 2020, vol 4, no. 1 management of urinary incontinence 63 southeastern european medical journal, 2020; 4(1) review article urinary incontinence: diagnostic evaluation and first-line treatment 1 ivan radoja 1, dunja degmečić* 2 1 department of urology, university hospital centre osijek, osijek, croatia, faculty of medicine, josip juraj strossmayer university of osijek 2 faculty of medicine, josip juraj strossmayer university of osijek, psychiatric clinic, university hospital centre osijek *corresponding author: dunja degmečić, ddegmecic@gmail.com received: feb 29, 2020; revised version accepted: apr 2, 2020; published: apr 27, 2020 keywords: urinary incontinence, cystoscopy, urodynamics, ultrasonography, parasympatholytics abstract urinary incontinence is defined as involuntary leakage of any amount of urine that negatively affects the individual's hygienic and social status. it is an important public health problem because it has a high prevalence and incidence. due to various social prejudices, it is often neglected and unreported. it is also characterized by its chronic appearance and complications such as decreased quality of life, sexual dysfunction and symptoms of anxiety and depression. the financial burden for symptomatic, conservative and surgical treatment of urinary incontinence is high. both women and men are reluctant to seek medical help, and they most often use coping strategies to deal with urinary incontinence symptoms, which include the use of incontinence aids such as adult diapers and pads. we conducted a systematic review of high-quality randomized controlled studies and of other review articles to compare circumstances surrounding the diagnostic evaluation and first-line treatment of urinary incontinence in women and men according to international continence society guidelines. because urinary incontinence can occur during any stage of life, diagnostic and therapeutic approach is different in women and men. social stigmatization created due to urinary incontinence may harm a person’s self-confidence and cause many negativities at a social level. timely recognition of the type and severity of symptoms of urinary incontinence is necessary to reduce the occurrence of the aforementioned complications. (radoja i, degmečić* d. urinary incontinence: diagnostic evaluation and first-line treatment. seemedj 2020; 4(1); 63-73) seemedj 2020, vol 4, no. 1 management of urinary incontinence 64 southeastern european medical journal, 2020; 4(1) introduction the international continence society (ics) has defined new terminology for lower urinary tract symptoms (luts), clinical signs and conditions. luts is now divided into symptoms occurring during storage of urine within the bladder (incontinence of urine, urgency, nocturia, nocturnal enuresis, overactive bladder syndrome, increased frequency of urination, nocturnal polyuria), symptoms during micturition (dysuria, abnormalities of urine flow) and symptoms after urination (feeling of incomplete bladder emptying, dripping of urine after urination) (1). the most common luts occurring in women is urinary incontinence (ui) and the most common luts in both sexes is overactive bladder syndrome (oab). ui is a symptom of involuntary leakage of urine subjectively reported by patients, or a clinical sign of involuntary leakage of urine objectively established by a physician (1). the amount of involuntary leakage of urine considered abnormal has not yet been defined, nor has the impact of ui on the psychological, physical and social functioning of the patient. the new definition specifies that ui is not a disease, but a symptom or clinical sign occurring due to various diseases of the neurological system, lower urinary tract, and muscles of the pelvic floor. diseases that can cause the onset of ui can be temporary and permanent, neurogenic or non-neurogenic (3). ui can manifest as an occasional involuntary leakage of a small amount urine or as the complete inability to retain urine within the bladder. given the complex aetiology of ui and the situations in which it occurs, ui is divided into several types. according to the ics, the most common types of ui are stress urinary incontinence (sui), urgency urinary incontinence (uui) and mixed urinary incontinence (mui) (1). sui is defined as involuntary leakage of urine during coughing, sneezing or another physical activity. it is most common in women, but it is also present in some men who underwent prostate cancer surgery. uui is defined as the observation of involuntary leakage from the urethral orifice associated with the individual reporting of the sensation of a sudden, compelling desire to void. unlike sui, this type of incontinence is unpredictable, it can happen at any time and it is independent of physical activity and bladder fullness. mui is characterized by an involuntary loss of urine associated with urgency and also on effort or physical exertion, including sporting activities, or on sneezing or coughing. other types of ui include postural ui, nocturnal enuresis, continuous ui, insensible ui and coital ui (4). ui is generally caused by the failure of pelvic floor muscles, the inability of the internal and external sphincter to withstand increased urinary bladder and abdominal pressure occurring during daily activities, or it can be a result of involuntary contractions of the bladder detrusor muscle. in most cases, women and men postpone seeking help from primary care health professionals. usually, this is because they believe that this is a normal condition occurring due to pregnancy and vaginal delivery in women, due to an enlarged prostate and complications of prostate surgery in men, or due to older age in both women and men. another reason is because they are not familiar with modern treatment possibilities. prevalence and etiology of urinary incontinence ui is estimated to affect more than 450 million people worldwide and is three times more common in women than in men, regardless of the type of ui (5). in the total adult population of both sexes in the united states of america (usa), the prevalence of ui ranges from 5% to 50%. ui can occur at any age, but the prevalence increases with age and affects approximately 40% of older women and approximately 15% of older men (6). currently, there are no data on the prevalence and incidence of ui in croatia, but it is probably similar to those from the usa. according to the latest research, the prevalence of ui in women and men of all ages, ranges from 20 to 50%, and the incidence of ui ranges from seemedj 2020, vol 4, no. 1 management of urinary incontinence 65 southeastern european medical journal, 2020; 4(1) 15 to 30% (7). the prevalence of ui increases with age and, therefore, amounts to 38% in patients over 60 years of age (8). the public health impact of ui is very high because the proportion of older adults in the total population of the usa and other developed western countries is increasing. according to recent scientific studies, sui is the most common type of ui, with prevalence ranging from 10 to 39% (9). mui is the next most common type of ui with a prevalence of 7% to 25% (10). uui occurs very rarely with a prevalence of 1% to 7% (10). uui occurs more frequently as part of oab. oab is defined as urinary urgency, usually accompanied by increased urination frequency during the day and increased urination frequency during sleep (i.e. nocturia), with or without uui, and in the absence of a urinary tract infection or another obvious disease of the urinary bladder (e.g. bladder cancer) (4). oab is often of idiopathic genesis or a consequence of a neurological disease, associated with excessive activity of the bladder detrusor muscle. the prevalence of oab ranges from 2% to 53% (11). normal total capacity of an adult bladder is approximately 400 to 500ml of urine. during the day, approximately 1400 ml of urine is produced, depending on the type and volume of the fluids we drink. the frequency of voiding is usually 2 to 3 times a day, and up to 1 time during the night. the normal desire for micturition occurs at a volume of 50% of normal total bladder capacity. some or most of these physiological functions and parameters are impaired in patients with ui, and are most commonly caused by the following: pelvic floor muscle disorders, bladder mucosa and detrusor muscle disorders, neurological diseases such as cerebrovascular stroke (cvs), multiple sclerosis (ms), dementia and parkinson's disease (12). ui can also occur in patients with psychological disorders such as schizophrenia, depression, and anxiety, or as a side effect of certain psychiatric medications (13). ui may also be associated with the onset of fecal incontinence, constipation and anorectal pain (14). neurological signals within multiple micturition centers and neural pathways (e.g. pontine micturition center, periaqueductal gray, pyramidal tracts), which control the response to changes in pressure within the bladder, are often insufficient in older age. this can result in the loss of voluntary and autonomous control of lower urinary tract functions (15). risk factors for sui in women are the number of childbirths, injuries to the organs and muscles of the pelvis during childbirth, episiotomies, children with high birth weight and hysterectomies (16). the occurrence of sui in men is associated with radical prostatectomy, because of the damage inflicted to the neural structures, the intrinsic urethral sphincter, and muscles and ligaments of the pelvic floor (12). indications for hysterectomy and prostatectomy can be various benign and malignant diseases of the uterus or prostate. annually, there are 170,000 new cases of prostate cancer and 60,000 new cases of uterine cancer in the usa (17, 18). in case of a localized prostate cancer, the therapeutic methods of choice are a radical prostatectomy or primary radiotherapy with curative intent. depending on the surgical method, experience, and skill of the surgeon, the incidence of ui after a radical prostatectomy can range up to 40% (19). in most cases, these men have sui, but they can also have mui with a previously unrecognized uui component (20). occurrence of uui or mui is very common in men and women after they undergo primary radiotherapy for prostate and uterine cancer. this is because the bladder mucosa is damaged by irradiation and becomes sensitive to changes in the volume and chemical composition of urine. the pathophysiology of sui includes damaged or weakened internal urethral sphincter of the bladder neck and pelvic floor muscles that support the bladder neck and urethra (16). pelvic floor muscles are composed of deep and superficial muscle fibers and connective fibers. they participate in a very complex anatomical and physiological relationship that maintains maximum urethral closure pressure in order to prevent leakage of urine. the relaxed bladder neck and pelvic floor muscles allow the bladder to empty. in women, the maximum urethral closure pressure and functional urethral length decrease with age. mechanisms that cause incontinence in older men are detrusor overactivity and internal sphincter insufficiency. in both sexes, bladder capacity and compliance seemedj 2020, vol 4, no. 1 management of urinary incontinence 66 southeastern european medical journal, 2020; 4(1) decrease with age, while excessive contractions of the bladder detrusor and residual urine simultaneously increase, which may cause ui (21). increased body mass index (bmi), family history of ui and older age are also associated with the onset of ui (22). as abdominal and bladder pressure increase, the pelvic floor muscles cannot tighten sufficiently enough to hold the urine within the bladder, and, therefore, the intravesical pressure overcomes the maximum urethral closure pressure resulting in urine leakage. neurological diseases such as dementia, cvs and ms, and bladder diseases such as chronic cystitis and cystocele, can all cause excessive detrusor contractions with consequent uui or hypermobility of the bladder neck and urethra with consequent sui (23). the prevalence of dementia in european countries ranges from 6 to 18% in people over 65, and from 30 to 50% in people over 85 (24, 25). in croatia, approximately 250 people per 100,000 inhabitants experience cvs annually (26). the prevalence of patients with ms in croatia is 143 patients per 100,000 inhabitants (27). some scientific studies predict an increase in the total number of cvs cases by as much as 34% in european countries in the period from 2015 to 2035, and they also predict an increase in the number of cvs survivors (28). the prevalence of parkinson's disease in the us is 572 patients per 100,000 inhabitants (29). cvs and parkinson's disease are more common in men, while dementia and ms are more common in women (30). diagnostic evaluation of urinary incontinence in croatia, family physicians and gynecologists play a leading role in the early identification of ui in primary care. basic diagnostic methods that can be implemented within primary care include medical history, bladder diary, urinalysis, plain radiography and ultrasonography of the urinary tract with a focus on post-void residual (pvr) urine. after a primary care assessment, women are referred to a urogynecologist and men are referred to a urologist. diagnostic evaluation of luts in urological and urogynaecological outpatient clinics includes detailed specific medical and sexual history, bladder diary, neurologic examination, prostate and genital examination in men, gynecological examination in women, and urethrocystoscopy. the causes of ui are obvious only in uncomplicated cases and a subsequently provided diagnosis is usually based on anamnestic data and a clinical examination. some cases, however, are very complex and, therefore, a thorough and extensive assessment of ui symptoms is required in order to provide a correct diagnosis and initiate appropriate treatment. during the evaluation, it is useful to perform a urinalysis, a urine culture test and a urethrocystoscopy. urinalysis and urine culture tests can detect urinary tract infections, hematuria, glycosuria, pyuria, crystalluria, and proteinuria. urethrocystoscopy is mandatory because it can confirm or exclude diseases such as bladder cancer or urolithiasis. if necessary, it is advisable to refer women and men with ui to a neurourology and urodynamics specialist for further diagnostic evaluation and treatment. urodynamics is defined as the measurement of physiological parameters relevant to the function of the lower urinary tract (1). urodynamics in women and men commonly involves uroflowmetry with a full bladder, but without a catheter, measurement of pvr, filling cystometry, a urethral pressure profile and pressure-flow studies (31). in complicated cases, such as post-prostatectomy incontinence and complications of transvaginal mesh surgery, a videourodynamic investigation with simultaneous measurement of urodynamic parameters must be performed, along with a radioscopy of the bladder filled with contrast medium in order to gain insight into the storage and micturition phase. sometimes it is difficult to explain the pathophysiology of luts only by correlating the patient's symptoms with the results of the urodynamic studies. additional diagnostic tools may include computed tomography (ct) of the upper and lower urinary tract, and magnetic resonance imaging (mri) of the brain and spinal cord for purposes of detecting tumors, aneurysms, injuries or other conditions. ct and seemedj 2020, vol 4, no. 1 management of urinary incontinence 67 southeastern european medical journal, 2020; 4(1) mri can also be used to monitor the activity and progression of the disease that caused the ui. treatment of urinary incontinence women and men are reluctant to seek professional help and talk about ui problems and related disorders such as sexual dysfunction, anxiety and depression. ui can cause a decrease in social interactions and physical activity, loss of self-esteem, depression, fear of involuntary leakage in society, avoidance of sexual relations and decreased productivity at work. in most cases, patients postpone going to the physician because they are not fully aware of the treatment options for ui, which can be very successful. as first-line treatment, physicians most commonly use various incontinence aids and strategies to cope with ui problems such as changing their lifestyle. if incontinence aids are not sufficiently effective, patients need to change multiple pieces of the same type of aid or combine different types of aids during a 24hour period. because of this, the costs of ui treatment and the financial burden on the healthcare system may be increased and the quality of life (qol) may be significantly reduced. in older women and men, the problems and costs of ui treatment are increasing. the world's fastest-growing elderly population includes persons aged 85 and over, and it will increase by as much as 12 times by 2025, while the number of people aged 100 and older will increase 15 times, i.e. from the current 210,000 to 3.2 million (32). in croatia, in 2001, there were 693,540 people older than 65 and 42,553 people older than 85 (33). depending on the underlying disease causing the ui and depending on the types of ui treatment, there are predictable costs of treatment (e.g. medication, surgery, and hospital stay costs) and unpredictable treatment costs (e.g. treatment of adverse drug effects and possible complications of surgery). larger share (50-75%) of ui treatment costs is attributed to body care products, laundry detergents, incontinence pads and diapers (34). the total annual cost of ui treatment in the us is very high and in 2000 it was estimated at 19.5 billion dollars (35, 36). the most recent estimate of total annual cost of ui treatment in the usa for all age groups was over 16 billion dollars, which is higher than the annual direct cost of treating breast, ovarian, and cervical cancers altogether (37, 38). according to one study, a person with more severe ui symptoms and a greater impact of ui on qol spends approximately 900 dollars annually for incontinence aids and body care products (39). another study found that the daily use of ui aids of one person with symptoms of ui results in an annual cost of 1825 dollars (approximately 150 dollars per month) (39). these differences may stem from the type and effectiveness of incontinence aids, primarily in terms of their power to absorb urine. the cost of treating one patient with uui or mui with antimuscarinic drugs alone, without absorbents, ranges from 95 to 290 dollars per month, depending on the type of drug (40). there are no data on the costs of treating ui in croatia, but we can assume that they are very similar to the previously mentioned data. in case of contraindications for surgery or pharmacotherapy, a symptomatic treatment that includes only incontinence aids that have a good overall effect can reduce treatment costs and improve the qol. depending on the type of ui, the choice of first-line treatment includes conservative methods such as: counselling about lifestyle changes (e.g. weight control, avoiding or reducing intake of alcoholic and other diuretic drinks, reducing fluid intake during the day and before bedtime), bladder training (keeping a bladder diary, delaying the need to urinate, increasing the amount of time between urination), use of incontinence aids (e.g. pads, diapers), pharmacotherapy (e.g. antimuscarinics, mirabegron), pelvic floor muscle exercises, biofeedback, extracorporeal magnetic innervation therapy and functional electrostimulation of pelvic floor muscles. treatment results of most conservative methods depend on the patient's motivation and co-operation, except in the case of incontinence seemedj 2020, vol 4, no. 1 management of urinary incontinence 68 southeastern european medical journal, 2020; 4(1) aids, such as diapers and pads, where treatment results depend on the absorption capacity and comfort. treatment of sui focuses on strengthening the supportive pelvic floor muscles by applying various conservative and surgical methods. the method of choice in uui treatment is the stabilization of detrusor overactivity by using pharmacotherapy. in the case of mui, it is necessary to treat the predominant symptomatology component. the most common surgical methods for sui treatment are the periurethral injection of bulking agents, insertion of pubovaginal slings and colposuspension. if pharmacotherapy proves to be inefficient in treating uui, either intravesical application of onabotulinum toxin a or a sacral neuromodulation is indicated. all of the above-mentioned surgical methods include higher costs when compared to conservative methods, and they also include treatment of complications and a number of days spent in the hospital. treatment of these complications may include the use of incontinence aids. some patients have absolute and relative contraindications and an increased risk of complications related to conservative and surgical treatments that are otherwise recommended according to the guidelines of the european association of urology (eau) and ics (41). these include diseases such as a recently experienced heart attack, blood clotting disorders, uncontrolled arterial hypertension, heart rhythm disturbances, the presence of metal implants and heart rhythm electro-stimulators, and glaucoma. these patients can be recommended to use incontinence aids, make lifestyle changes, train their bladder, and do pelvic floor muscle exercises as part of the prescribed therapeutic method in order to help maximize the therapeutic effect. incontinence aids can also improve the qol of patients until a proper diagnosis of luts is made and treatment initiated. they can also be a permanent solution for patients who have an increased risk of developing complications during the implementation of diagnostic and therapeutic methods related to ui. achieving effective and discreet urinary retention with the use of incontinence aids is one of their most important features and it helps improve the users’ qol (42). the most common products used for ui symptoms in men and women are incontinence pads and diapers. they absorb urine after one or more episodes of involuntary urine leakage, depending on their power of absorption. these products protect against leakage, odour, and prevent moisture from getting onto the genital area and skin. other products that can be used with ui patients include waterproof bed sheets and urinary condoms. waterproof bed sheets can be washed in the washing machine and reused, but they can also be disposable. they are placed under normal bed sheets and above the mattresses, and they absorb urine. incontinence pads and diapers have a hydrophobic layer that absorbs urine below the surface, allowing the genital skin to remain dry. they are usually a temporary measure for improving the qol, as they provide safety and confidence during the day and help maintain a normal social life. tampons are not recommended for use in women with ui, but they can be useful in situations, such as exercise, as their position can affect bladder neck lifting. this is due to the very close anatomical relationship of the vagina and bladder neck. menstrual pads should also be avoided when dealing with ui issues. a lot of women use menstrual pads instead of incontinence pads. however, they do not have the same technology and, therefore, these pads remain wet and can cause chronic skin changes, contact dermatitis, and fungal infections. in patients experiencing involuntary leakage of smaller amounts of urine, incontinence pads are more comfortable to wear. in patients experiencing involuntary leakage of moderate to large amounts of urine, which occur several times a day, it is usually necessary to use diapers with a greater absorption capacity, in comparison to that of incontinence pads, or use a combination of both types of supplies. for practical reasons, mostly due to their shape and size, incontinence pads are mostly used by mobile patients who can go to the toilet on their own. unlike diapers, pads are more discreet as they are not noticeable under clothing and are seemedj 2020, vol 4, no. 1 management of urinary incontinence 69 southeastern european medical journal, 2020; 4(1) easy to put on and remove. in most cases, diapers are used by immobile patients, with or without cognitive impairment, who cannot go to the toilet on their own and who do not need to change supplies frequently. diapers can absorb greater amounts of urine, but because they need to be fixated with adhesive tapes, they are not practical for removing and repositioning during bladder training or for personal hygiene related reasons. incontinence pads are more convenient to remove and reuse during bladder training, but they do not have the same absorption power as diapers, meaning that more incontinence pads need to be used during the day. discussion according to eau guidelines and world health organisation guidelines on integrated care for older people (who icope), first-line of treatment for ui symptoms includes lifestyle changes, bladder training, pelvic floor exercises and incontinence aids (43-45). with the help of incontinence aids, it is possible to prevent involuntary leakage on clothing, eliminate the unpleasant odour of urine and reduce the possibility of bacterial infections and chronic dermatitis on the mucous membrane and skin of the genital region. the use of these aids is very important in patients who cannot undergo active treatment by other conservative or surgical methods due to contraindications, and in patients who refuse active treatment. it should be noted that, in most cases, these aids are used in patients who have uui or mui with a predominant uui component due to the different aetiology of these types of ui. because the onset of sui symptoms is predictable, various behavioural strategies, such as bladder emptying before a physical activity, can help avoid episodes of involuntary urine leakage, and, consequently, the use of these aids is minimized. uui is unpredictable because an overactivity of the bladder detrusor can occur at any time and, therefore, implementing similar strategies does not have a positive effect. one of the strategies used by patients with uui is the mapping of toilets, meaning that they keep a record on the locations of toilets in a city, shopping centres, and public institutions. uui patients are advised to change their lifestyle, train their bladder and use incontinence aids. pharmacotherapy with antimuscarinic and betaagonist mirabegron is also indicated as first-line therapy in patients with uui. some patients may have contraindications for pharmacotherapy. contraindications to antimuscarinic pharmacotherapy include acute or chronic urinary retention, digestive system diseases (e.g. toxic megacolon), myasthenia gravis, narrowangle glaucoma, renal failure requiring haemodialysis and severe impairment of liver functions. contraindications for pharmacotherapy with mirabegron include severe uncontrolled hypertension, defined as systolic blood pressure ≥ 180 mmhg and diastolic blood pressure ≥ 110 mmhg. if there are any contraindications for pharmacotherapy, patients will be provided with incontinence aids. they will also have to change their habits, train their bladder, and implement other behavioural strategies. the prevalence of neurological diseases and malignant diseases of the genitourinary system is high, and they usually include ui as a consequence. thanks to modern diagnostic and therapeutic options, the qol of these patients has greatly improved and their life prolonged. as a result, there are more and more patients with some degree of disability and symptoms of ui. in these patients, a specific approach to treatment and rehabilitation is required in order to maintain their cognitive and motor skills and help them control the ui symptoms. neurological diseases can cause immobility or impaired mobility, depending on their degree and the level of neurological damage, and this can cause complications such as deep vein thrombosis and decubital ulcers (44). bladder function in these patients is often neglected, as is the fact that they often have symptoms of ui. it is very important that verticalization and mobility of these patients be restored in a timely manner through daily bladder training. it is also important to restore their bladder capacity and increase the muscle strength of detrusors, and also to prevent other complications. if patients are immobile and spend most of their 24 hours seemedj 2020, vol 4, no. 1 management of urinary incontinence 70 southeastern european medical journal, 2020; 4(1) in a supine or sitting position, the sensory and motor functions of the bladder may deteriorate. when training the bladder, patients are advised to urinate during the day while retaining urine to a certain extent before going to the toilet. if patients have ui, they must use certain types of aids that will hold the urine until they go to the toilet. diapers would be the most appropriate choice because they have a greater capacity to absorb urine in comparison to incontinence pads. a negative feature of diapers is that they are not as discreet as pads, because they are larger and noticeable under clothing. this can cause fear of an unpleasant odour of urine, social prejudice, anxiety, depression, decreased qol and social stigmatization (45). incontinence pads are more discreet than diapers, but because of their lower absorption capacity, patients usually change up to 7 pads a day (45). this inflicts a huge financial burden on the health system. ui aids of an adequate type and size are required in order to prevent the detection of ui symptoms in social situations. this is achieved by reducing the absorption effect of episodes and the volume of involuntary leakage of urine during 24 hours. in addition, they also help reduce the possibility of complications, such as skin and genital mucosal damage. all patients with ui use some kind of an incontinence aid, regardless of the type and severity of ui symptoms. there are significant differences in design, absorption capabilities and other features between ui aids. there are also significant individual differences in personal attitudes of patients with ui and in their desires for incontinence treatment. some aids are better designed for men than for women, for example, some aids have better absorption capabilities. depending on the type and duration of physical and social activities, ui patients usually combine several types of aids and use multiple pieces of aids, especially incontinence pads, over a 24hourperiod. reducing the financial burden on the healthcare system could be achieved by providing patients with better-designed ui aids suitable for different circumstances and activities for 24-hour periods. with the help of these conservative first-line treatment methods, patients can maintain an active lifestyle and improve sexual function and their qol (46, 47). ui is a multidisciplinary problem and all physicians involved in the care of people with ui need to be well informed about the new scientific research data regarding diagnosing and treating of ui, as well as of other luts. conclusion with the help of primary prevention measures, women and men should be educated and encouraged to seek help from a family physician, gynaecologist or urologist if they have ui symptoms. after reviewing the patient's medical history, and performing a physical exam, these healthcare professionals can determine the type of ui and begin appropriate treatment, if possible. a three-day bladder diary and various screening questionnaires, such as international consultation of incontinence questionnaire – short form and king's health questionnaire, are useful in the evaluation of the type and severity of ui symptoms. sometimes it is very difficult to identify the type of ui and it is, therefore, necessary to refer the patient to a neurourologist for a urodynamic assessment. there are many conservative and surgical treatment methods for ui. in most patients, we can achieve full continence status or a major improvement in alleviating ui symptoms so that a normal lifestyle can be maintained. there are many aids that can be prescribed to patients who have symptoms of ui, such as incontinence pads and diapers, which have different urine absorption properties. ui aids can improve the qol until proper treatment is initiated, or they can be a permanent solution in patients with contraindications for pharmacological or surgical treatment. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. . seemedj 2020, vol 4, no. 1 stigmatization of patients with viral infections and mass psychogenic illness 71 southeastern european medical journal, 2020; 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5(1) review article the endemicity of lassa fever in west africa; appropriate mitigative measures 1 israel oluwasegun ayenigbara 1*, george omoniyi ayenigbara 2, michael olubodun abulogbon 1, olayemi febisola laleye 3, bolarinwa akinyemi 2 1 school and community health education unit, department of health education, university of ibadan, ibadan, nigeria 2 department of human kinetics and health education, adekunle ajasin university, akungba-akoko, ondo state, nigeria 3 health and safety education unit, department of health education, university of ibadan, ibadan, nigeria *corresponding author: israel oluwasegun ayenigbara, histrealite2647@gmail.com received: may 10, 2020; revised version accepted: jan 15 2021; published: apr 28, 2021 keywords: lassa fever, zoonoses, west africa, prevention, public health abstract lassa fever is an ancient disease and is endemic in most west african countries. importantly, lassa fever is a dangerous and virulent disease because it exerts deleterious effects on many vital organs in the body. due to its endemic nature and the yearly occurrence of the disease in terms of infection and mortality cases in some west african countries, specifically nigeria, there is need to reexamine and reemphasize viable prevention alternatives. on this backdrop, this review provides a broad overview of lassa fever, with the main emphasis on preventive measures. infection with the lassa fever virus has severe consequences on health; in this respect, multifaceted preventive measures that ensure and guarantee no contact with multimammate rodents should be adopted. furthermore, contact with the feces and urine of multimammate rats should be avoided, personal hygiene should always be practiced, environmental sanitation should be ensured and carried out often, the consumption and eating of rats should be discouraged, abolished and ultimately stopped, and food containers should always be kept tight and closed. (ayenigbara io, ayenigbara go, abulogbon mo, laleye of, akinyemi b. the endemicity of lassa fever in west africa; appropriate mitigative measures. seemedj 2021; 5(1); 25-36) mailto:histrealite2647@gmail.com seemedj 2021, vol 5, no. 1 lassa fever in west africa 26 southeastern european medical journal, 2021; 5(1) introduction lassa fever develops through the infection by the lassa virus (lasv) (1, 2). although the disease was first discovered in the early 1950s, the causative agent was not recognized or known until the late 1960s. the virus that causes the disease belongs to the family of arenaviridae viruses (3). the history of lassa fever dates back to the 1950s, when the first case was recorded in nigeria, borno state, in the town of lassa, after which the disease was named lassa fever in 1969 (3). presently, the disease is endemic in west african countries because of the yearly cases of infection and death (1, 4-6). numerical estimates indicate that confirmed cases of lassa fever range between 350000 and 550000, and over 5000 deaths per year are recorded globally (3). hosts of the lassa virus are infected multimammate rats and rodents, and humans usually become infected with the virus due to contact with urine or feces of infected animals (3). the issue of lassa fever is a yearly phenomenon in nigeria, and the surge of infections in humans is usually seen in the dry season between december and april, due to the reproduction cycle of new multimammate rats and rodents in the rainy season (7). the lassa virus is easily transmitted and highly contagious, and the disease it causes is deleterious because it has severe health consequences for the whole system (3). once a person is infected with the lassa virus, the virus weakens organs in the body, consequently resulting in organ malfunctions and depletion of the whole system. in regard to its public health importance, lassa fever is a yearly phenomenon in endemic countries because of the rapid multiplication of the virus in the newly infected multimammate rats and rodents, which, once infected, continue to shed the virus in their feces and urine throughout their life (7). although lassa fever may not be completely eradicated due to peculiar reasons and lack of existing vaccines against the causative virus, transmission of the lassa virus could however be diminished and absolutely prevented. hence, this synopsis presents detailed preventive measures against lassa fever. sources of information the review focuses on workable prevention alternatives against lassa fever. the literature review was purposely selected with keywords on the topic of discourse from pubmed, google scholar and springerlink databases. all eligible studies included are clinical trials, metaanalyses, randomized controlled trials, systematic and review articles on the topic of discourse. furthermore, major international and national health agencies’ databases were searched to find valuable information for the review. biological description of the lassa virus lassa viruses are typically enclosed, singlestranded, bisegmented and ambisense rna in nature (8). their genome is contained in two rna segments that code for two proteins each, one in each sense, for a total of four viral proteins. the large segment encodes a small zinc finger protein (z), which regulates transcription and replication, and the rna polymerase (l) (9). the small segment encodes the nucleoprotein (np) and the surface glycoprotein precursor (gp) or the viral spike, which is proteolytically cleaved into the envelope glycoproteins gp1 and gp2 that bind to the alpha-dystroglycan receptor and mediate host cell entry (10). lassa fever causes hemorrhagic fever frequently shown by immunosuppression. the lassa virus replicates very rapidly and demonstrates temporal control in replication. the first replication step is transcription of mrna copies of the negativeor minus-sense genome. this ensures an adequate supply of viral proteins for subsequent steps of replication, as the np and l proteins are translated from the mrna (9, 10). the positiveor plus-sense genome then makes viral complementary rna (vcrna) copies of itself. in addition, the rna copies are a template for producing negativesense progeny, but mrna is also synthesized seemedj 2021, vol 5, no. 1 lassa fever in west africa 27 southeastern european medical journal, 2021; 5(1) from it. the mrna synthesized from vcrna is translated to make the gp and z proteins (9, 10). this temporal control allows the spike proteins to be produced last, and therefore delay recognition by the host immune system. nucleotide studies of the genome have shown that lassa fever has four lineages: three found in nigeria and the fourth in guinea, liberia, and sierra leone (11). the nigerian strains seem likely to have been ancestral to the others, but further research is needed to substantiate this (11). the lassa virus enters the host cell through the cell-surface receptor, the alpha-dystroglycan (alpha-dg), which is a versatile receptor for proteins of the extracellular matrix (10). it shares this receptor with the prototypical old world arenavirus, the lymphocytic choriomeningitis virus. receptor recognition depends on specific sugar modification of alpha-dystroglycan by a group of glycosyltransferases known as the large proteins (10). specific variants of the genes encoding these proteins appear to be under positive selection in west africa, where lassa fever is prominent (12). alphadystroglycan is also used as a receptor by viruses of the new world clade carenaviruses (oliveros and latino viruses). in contrast, the new world arenaviruses of clades a and b, which include the important viruses machupo, guanarito, junin, and sabia, in addition to the non-pathogenic amapari virus, use the transferrin receptor 1 (12). a small aliphatic amino acid at the gp1 glycoprotein amino acid position 260 is required for high-affinity binding to alphadg. likewise, gp1 amino acid position 259 also appears to be important, since all arenaviruses showing high-affinity alpha-dg binding possess a bulky aromatic amino acid (tyrosine or phenylalanine) at this position (10, 12). unlike most enveloped viruses, which use clathrincoated pits for cellular entry and bind to their receptors in a ph dependent fashion, lassa and lymphocytic choriomeningitis virus instead use an endocytotic pathway independent of clathrin, caveolin, dynamin and actin (12). once they enter the cell, the viruses are rapidly delivered to endosomes via vesicular trafficking, albeit one that is largely independent of the small gtpases rab5 and rab7. on contact with the endosome ph-dependent membrane, fusion occurs and is mediated by the envelope glycoprotein, which at the lower ph of the endosome binds the lysosome protein lamp1, which results in membrane fusion and escape from the endosome (12). the lifecycle of the lassa virus is similar to the old world arenaviruses. the lassa virus enters the cell by receptor-mediated endocytosis. the specific endocytotic pathway used is still unknown, but cellular entry is sensitive to cholesterol depletion (13). the receptor used for cell entry is alpha-dystroglycan, a highly conserved and ubiquitously expressed cell surface receptor for extracellular matrix proteins. dystroglycan, which is later cleaved into alpha-dystroglycan and beta-dystroglycan, is originally expressed in most cells to mature tissues, and it provides a molecular link between the ecm and the actin-based cytoskeleton (13). after the virus enters the cell by alphadystroglycan-mediated endocytosis, the lowph environment triggers ph-dependent membrane fusion and releases the rnp (viral ribonucleoprotein) complex into the cytoplasm. viral rna is unpacked, and replication and transcription commence in the cytoplasm (13). as replication starts, both s and l rna genomes synthesize the antigenomic s and l rnas, and from the antigenomic rnas, genomic s and l rna are synthesized. both genomic and antigenomic rnas are needed for transcription and translation. the s rna encodes gp and np (viral nucleocapsid protein) proteins, while l rna encodes z and l proteins. the l protein usually represents the viral rna-dependent rna polymerase (14). when the cell is infected by the virus, l polymerase is associated with the viral rnp and initiates the transcription of the genomic rna. the 5’ and 3’ terminal 19 nt viral promoter regions of both rna segments are necessary for recognition and binding of the viral polymerase. primary transcription first transcribes mrnas from the genomic s and l rnas, which code np and l proteins, respectively (14). transcription terminates at the stem-loop (sl) structure within the intergenomic region. arenaviruses use a cap-snatching seemedj 2021, vol 5, no. 1 lassa fever in west africa 28 southeastern european medical journal, 2021; 5(1) strategy to gain the cap structures from the cellular mrnas, which is mediated by the endonuclease activity of the l polymerase and the cap-binding activity of np. antigenomic rna transcribes viral genes gpc and z, encoded in genomic orientation, from s and l segments, respectively. antigenomic rna also serves as the template for replication (15). after translation of gpc, it is post-translationally modified in the endoplasmic reticulum. gpc is cleaved into gp1 and gp2 at the later stage of the secretory pathway. cellular protease ski-1/s1p is responsible for this cleavage (14). the cleaved glycoproteins are incorporated into the virion envelope with the virus buds and release from the cell membrane (14). brief pathogenesis of lassa fever lassa fever is mostly caught by humans through exposure to urine or feces of the host rodents, commonly through the contamination of uncovered food items at home (3). additionally, the spread of the lassa fever could occur through direct contact between infected humans. people who live in overcrowded areas or environments where the host rodents are abundant, as well as places with a lack of standard hygienic measures, are at a higher risk of infection with the lassa virus. furthermore, human-to-human route of lassa fever transmission has been confirmed, which correlates with infection cases in healthcare workers treating lassa fever patients. likewise, family members caring for infected relatives can be infected with lassa fever through the humanto-human route of transmission (3, 16). presently, there is no empirical data about viral shielding in human breast milk due to increased and elevated viremia (17). symptoms of the disease include a flu-like illness characterized by fever, general weakness, cough, sore throat, headache, and gastrointestinal manifestations. hemorrhagic manifestations include vascular permeability (15). upon entry into the body, the lassa virus infects almost every tissue in the human body. it starts with the mucosa, intestines, lungs and the urinary system, and then progresses to the vascular system (18). the main targets of the virus are antigen-presenting cells, mainly dendritic and endothelial cells (19). generally, when a pathogen enters a host, the innate defense system recognizes the pathogenassociated molecular patterns (pamp) and activates an immune response (19). one of the mechanisms detects double-stranded rna (dsrna), which is only synthesized by negativesense viruses (19). in the cytoplasm, dsrna receptors, such as rig-i (retinoic acid-inducible gene i) and mda-5 (melanoma differentiationassociated gene 5), detect dsrnas and initiate signaling pathways that translocate irf-3 (interferon regulatory factor 3) and other transcription factors to the nucleus (20). translocated transcription factors activate the expression of interferons, and these initiate adaptive immunity. np encoded in the lassa virus is essential in viral replication and transcription, but it also suppresses the host innate ifn response by inhibiting the translocation of irf-3 (20). np of the lassa virus is reported to have an exonuclease activity to only dsrnas (20). the np dsrna exonuclease activity counteracts ifn responses by digesting the pamps, thus allowing the virus to evade host immune responses (21). diagnosis of lassa fever accurate and rapid diagnosis of lassa fever is especially challenging because of unclear signs and symptoms, different variants of the lassa virus present in west africa, and laboratory safety concerns regarding highly virulent pathogens (22). viral culture remains the “best standard” for lassa fever diagnosis across the diverse lassa strains, but requires a clinically nonactionable amount of time and safety level4 precautions to perform (22). likewise, nucleic acid-based assays have become the clinical diagnostic standard and may be performed rapidly on inactivated specimens under safety level-2 conditions, but may have false-negative results due to the high variance among viruses (22). viral antigen assays may provide a rapid diagnosis early on during the illness, but may miss the diagnosis at later stages, once the antigenemia phase has resolved (22). the detection of a new igm antibody response can seemedj 2021, vol 5, no. 1 lassa fever in west africa 29 southeastern european medical journal, 2021; 5(1) diagnose lassa fever, but may miss the diagnosis during the early stage of illness, may be falsely negative in severe infections where patients are unable to mount a serological response, and may remain positive for a prolonged period, potentially leading to falsepositive results (22). a rise in baseline antibody titers between acuteand convalescent-phase serum or a positive igm accompanied by the development of a new positive igg response may be more indicative of acute lassa fever in regions where it is endemic than a single positive igm titer (22). to sum up, an orthogonal diagnostic system employing molecular (pcr-based) and immunological (antibody-based) assays provides the greatest confidence in a diagnostic result for lassa fever (23). also, rapid diagnostic tests (rdts) such as lateral flow immunoassays (lfis) can be an important addition to the orthogonal system and can significantly reduce infections in ongoing outbreaks and in endemic areas (23). furthermore, multiplex, magnetic bead-based assays are a significant improvement over traditional enzyme-linked immunosorbent assays (elisas) (23). clinical manifestations of lassa fever the period between contact with the lassa virus and the appearance of signs and symptoms is usually 6 to 20 days (3). there are numerous signs and symptoms of lassa fever, however, general weakness, migraine and headache, sore throat, muscle pain, chest pain, nausea, vomiting, diarrhea, cough, and abdominal pain are most commonly reported and seen in patients (3, 24). in addition, protein may be seen in the urine of lassa fever patients, and patients may also experience shock and seizures, while coma could occur in advanced stages of the disease due to the absence of treatment (3, 24). although other infectious diseases may present with the aforementioned signs and symptoms, it is imperative to visit a hospital or a healthcare clinic for proper diagnosis and early treatment of any diagnosed infection. about 25% of lassa fever survivors have hearing problems, which are typically reversible (25, 26). incidences of hair loss during recuperation from lassa fever have been reported, and death often occurs within a few days in severe cases of the disease without treatment. clinical presentation of lassa fever is serious in pregnancy, especially in the last stage of pregnancy, because the disease usually causes miscarriages and stillbirths due to the strong affinity of the lassa virus for fetal tissue (3). in addition, once a person is infected with the lassa virus, the virus weakens the organs in the body and causes organ malfunctions and depletion of the entire system (17, 24). the epidemiological trend of lassa fever the lassa virus was first identified in the late 1960s by a healthcare worker in a healthcare center in lassa, a town in nigeria (4). after diagnosis, the contagious lassa virus was confirmed (27). over the years, lassa fever has spread from the shores of nigeria, and numerous west african countries, such as burkina faso, ghana, togo, ivory coast, benin, liberia, guinea and mali, are either lassa feverendemic or have had cases of infection (28-31, table 1). the infection rate of lassa fever is high based on geographic location (i.e. 1.9% in developed countries compared to 56% in developing countries); likewise, lassa fever caught in hospitals results in more deaths (32, 33). furthermore, findings from hospital-based serosurveillance related with suspected cases of lassa virus infection revealed that basic hygiene measures among medical workers resulted in fewer infection cases compared to local inhabitants in villages, where basic hygiene practices are less common (33). the transmission rate of lassa fever depends on the geographic location, with higher frequencies in developing countries compared to developed countries (34). seemedj 2021, vol 5, no. 1 lassa fever in west africa 30 southeastern european medical journal, 2021; 5(1) table 1. summary of west african nations with lassa fever cases, 1962-2018. adapted from: (3) s/n west african countries with at least one case of lassa virus infection endemic west african countries with lassa fever west african countries reporting lassa fever outbreaks 1 mali nigeria nigeria 2 burkina faso guinea guinea 3 ghana liberia liberia 4 togo sierra leone sierra leone 5 benin 6 ivory coast lassa fever in nigeria as the world battles the current covid-19 pandemic, nigeria faces a double battle with two highly contagious viral infections. many years after it was discovered, lassa fever is still a major public health problem in nigeria (35). the aforementioned statement is evidenced by the massive outbreak of the disease in 2018 in the country, where 18 out of the 36 states of the federation were affected by lassa fever; this was the biggest and the worst case of the disease in history (36, 37). in this respect, the nigerian health authority declared the disease an emergency (38). from the beginning of 2020 until 26th december, 6732 associated cases of lassa fever were accounted for in nigeria, and 1181 samples returned positive from laboratory reports (39)d. furthermore, from the beginning of 2020 until the end of 2020, 244 deaths occurred in 27 states, and 131 local government areas were affected by lassa fever across the federation, while the general case fatality rate (cfr) for 2020 was 20.7% (39). numerical estimates also revealed that edo, ondo and ebonyi states had altogether 75% of the positive cases of lassa fever in nigeria for 2020 (39). cases of lassa fever were evident in virtually all age groups; however, the predominant age range of people with confirmed infection for 2020 was 21-30, while the gender ratio for male and female was 1:0.9 (39). due to the overburdening effects of the coronavirus disease pandemic (covid-19) on the healthcare system, lassa fever also exerted additional effects on health practitioners, because many health workers were infected with both diseases mistakenly, with more than two deaths recorded due to lassa fever in 2020 (7). lassa fever patients were treated and managed in various hospitals and healthcare centers across the country, secondary contacts were also identified, and follow-ups were done to ensure proper tracking and monitoring of the disease (39). lassa fever is predominant in nigeria and the onset of the disease is usually between december and june every year. imperatively, the seasonal surge in new lassa fever infection cases and deaths in the whole of nigeria is a cause for concern and ought to be observed intently by both national and international health authorities (38). in addition, bordering countries, for example togo and benin, have had imported cases of lassa fever from nigeria in the past; hence, bordering countries should be closely monitored as well (38). main emphasis on the prevention of lassa fever there are no protective vaccines against lassa fever as of 2020 (40). because of the endemic nature of lassa fever, the disease was listed among future causes of disease outbreaks by the world health organization (41, 42). lassa fever is prevalent in most west african countries and results in the deaths of thousands of people ever year (3). due to the exponential rise in seemedj 2021, vol 5, no. 1 lassa fever in west africa 31 southeastern european medical journal, 2021; 5(1) mortality and infection cases of lassa fever in the west africa subregion, especially nigeria, it is imperative to advance and reemphasize knowledge about prevention of the disease. prevention at home the transmission route of the lassa virus to the secondary host (humans) should be forestalled, hindered and disrupted by limiting and reducing any exposure or contact with the feces and urine of the primary host (rodents) in lassa fever hotspot areas and geographic locations, because worthwhile and beneficial efforts regarding the prevention of the disease depend on sustainable, adequate and proper personal and community hygiene efforts (43, figure 1). furthermore, successful and effective preventive measures against lassa fever in the home include keeping edible items in closed, tight and clean containers, discarding, disposing of or burning waste and refuse far away from residential areas, basic personal hygiene practices such as washing hands with clean running water and soap before handling any edible items, immediate cleaning and washing of used plates and pots with detergent and clean water, tidy arrangement of kitchen utensils and other materials in clean and well-ventilated cabinets, always keeping kitchen floors clean and tidy, and proper disposal of leftover foods. in addition, prompt caution and preventive measures should be adopted by relatives when caring for persons with lassa fever (3).. figure 1. prevention of lassa fever at home (self-developed) seemedj 2021, vol 5, no. 1 lassa fever in west africa 32 southeastern european medical journal, 2021; 5(1) prevention at hospitals high safety measures must always be adopted by all healthcare workers in different hospitals, clinics and healthcare centers, even when the diagnosis and cause of infection are unknown. preventive and control safety measures include essential hand washing with soap, detergent or antiseptic and clean running water, respiratory hygiene and the appropriate use of personal protective equipment (ppe) (3). furthermore, all rules and guidelines set by the world health organization for dealing with cases of highly infectious diseases should be strictly followed by healthcare workers treating lassa fever patients. in addition, proper checking of personal protective equipment (ppe) should be ensured and the equipment should always be made available to healthcare workers in lassa feverprominent areas, such as nigeria and other neighboring countries. likewise, laboratory workers are at a higher risk of lassa virus infection; hence, all lassa virus samples in the laboratory should be handled with extreme care and caution. in addition, the infection prevention and control authorities (ipc) should ensure that healthcare centers and laboratories meet the requirements for operation, especially in lassa fever hotspot areas (44). it is imperative to ascertain that all healthcare centers attending to lassa fever cases operate with the standard achievable around the world, and routine ipc training should also be made mandatory for all healthcare workers handling cases of lassa fever (44). prevention among travelers migrants moving from countries where lassa fever is prominent often transfer and import the disease to other locations or regions; this is evident in togo and benin, since some of the reported cases in the two countries were imported from nigeria (38). strict and continuous border checking and inspection of medical reports of travelers is paramount. imperatively, all border rules and regulations pertaining to infectious disease checking during border crossing should be strictly enforced by border patrols and healthcare workers stationed at border crossings. likewise, persons visiting lassa fever-endemic nations for vacation should visit the respective embassies and consulates in the countries they visit in order to get information on lassa fever-prone areas in such countries, as well as to get general health information on other infectious diseases prevalent in such countries. furthermore, febrile migrants who recently returned from lassa fever-predominant areas should urgently visit a healthcare clinic or hospital in their current location in order to get a proper diagnosis and early treatment (45). treatment options for lassa fever treatment options for lassa fever are few and treatments are mainly symptomatic (8). management of bleeding and hydration is important, particularly in hemorrhagic cases. likewise, pain management through the use of opiates is prescribed (8). due to the permeability of blood vessels, pulmonary edema is a concern, and fluid infusion must therefore be carefully monitored (8). an antiviral drug (ribavirin) offers beneficial effects for lassa fever patients with poor prognoses, and is usually reserved for patients with higher herliver enzyme levels (ast value > 150) (8). even though it is a drug with significant side effects, ribavirin is the drug of choice in many cases of lassa fever (8, 46). recommendations based on this study, the following recommendations are made: a) contact with multimammate rats, especially their feces and urine, should be avoided. b) personal hygiene, such as hand washing with clean running water and antiseptic soap, should be practiced at all times. seemedj 2021, vol 5, no. 1 lassa fever in west africa 33 southeastern european medical journal, 2021; 5(1) c) environmental sanitation, such as clearing and cleaning of the environment, should be done frequently in order to destroy the habitats of multimammate rats. d) the barbaric act of eating rats should be discouraged, abolished and ultimately stopped. e) containers for food items should be kept tight and closed at all times to prevent rats from accessing them, and any food items with traces of rat feces or urine should be discarded immediately. f) healthcare workers, especially nurses, doctors and laboratory scientists, should follow the basic aseptic practices when treating patients infected with lassa fever. g) health education on lassa fever should be intensified for everyone. h) the rearing and breading of cats at home should be encouraged. this would help reduce the population of rats in the environment, especially in rural areas. conclusions lassa virus is highly infectious and could infect any individual, since no one is immune to the disease. lassa fever is endemic in most west african countries, with yearly cases in terms of infection and mortality in nigeria. people at severe risk of infection with lassa fever are individuals living in unhygienic and dirty environments with overcrowded living conditions, because these are the scenarios and situations in numerous lassa fever-endemic areas. health consequences caused by the infectious lassa fever are enormous. in this respect, multifaceted preventive measures that ensure, encourage and guarantee no contact with multimammate rodents and rats should be adopted. furthermore, there should be a strong synergy and cooperation between governments and border patrol officers of lassa feverendemic areas to work out effective ways to minimize the rate of cross-border transmissions of the disease. likewise, major international health organizations should help and assist with public health experts and personnel, logistic and vital information in curtailing the spread of lassa fever in developing countries. acknowledgement. the authors appreciate all researchers working on the epidemiology of lassa fever and its prevention who contributed to the ideas discussed in this article. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. oloniniyi ok, unigwe us, okada s, kimura m, koyano s, miyazaki y, iroezindu mo, ajayi na, chukwubike cm, chika-igwenyi nm, ndu ac, nwidi du, abe h, urata s, kurosaki y, yasuda j. genetic characterization of lassa virus strains isolated from 2012 to 2016 in southeastern nigeria. plos negl trop dis. 2018; 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5(1) 43. center for disease control and prevention. lassa fever prevention. 2014. https://www.cdc.gov/vhf/lassa/prevention/in dex.html. retrieved 10th may, 2020. 44. ijarotimi it, ilesanmi os, aderinwale a, abiodun-adewusi o, okon i-m. knowledge of lassa fever and use of infection prevention and control facilities among health care workers during lassa fever outbreak in ondo state, nigeria. pan afr med j. 2018;30:56. doi:10.11604/pamj.2018.30.56.13125. 45. kofman a, choi mj, rollin pe. lassa fever in travelers from west africa, 1969-2016. emerg infect dis. 2019; 25(2):245-248. doi: 10.3201/eid2502.180836. 46. eberhardt ka, mischlinger j, jordan s, groger m, günther s, ramharter m. ribavirin for the treatment of lassa fever: a systematic review and meta-analysis. int j infect dis. 2019; 87:15-20. doi:10.1016/j.ijid.2019.07.015.. 1 author contribution. ioa led the conception and writing of the paper. moa, goa, ba and ofl helped with the literature search, reviewed all tittles, abstracts related to the topic, and full text. ioa and moa assisted with the recommendations in writings. ofl helped with reference checking and completion, while goa and ioa helped with the editing and proofreading of the whole manuscript. all the authors read and agreed to the final copy of the manuscript for publication. seemedj 2020, vol 4, no. 2 croatian translation of sahlsa-50 121 southeastern european medical journal, 2020; 4(2) original article croatian translation and linguistic adaptation of the short assessment of health literacy for spanish adults (sahlsa-50) 1 terezija berlančić 1,2,3, marin kuharić 4, dragan janković 5, jakov milić 1, katarina badak 1, lada zibar *1,6 1 faculty of medicine osijek, josip juraj strossmayer university of osijek, croatia 2 department of gynecology and obstetrics, county general hospital našice, croatia 3 faculty of economics in osijek, josip juraj strossmayer university of osijek, croatia 4 faculty of dental medicine and health osijek, josip juraj strossmayer university of osijek, croatia 5 department of neurosurgery, university hospital of marburg, germany 6 department of nephrology, clinical hospital merkur, zagreb, croatia *corresponding author: lada zibar, ladazibar@gmail.com received: mar 1, 2020; revised version accepted: jul 16, 2020; published: nov 12, 2020 keywords: croatian language, health literacy, sahlsa-50, translation abstract introduction: population needs health literacy (hl) in order to understand health-related topics. short assessment of health literacy for spanish adults (sahlsa-50) could be a high-quality tool for assessing hl in adults. unlike questionnaires in the english language, which are not easily modifiable for croatian, translation of sahlsa-50 from spanish into croatian could be an appropriate choice. there are no similar questionnaires to assess hl in croatian to date. aim: to translate sahlsa-50 into croatian using a validated procedure and to adapt it linguistically, so that it could be used in the croatian-speaking area to measure hl in the croatian population. material and methods: sahlsa was developed for the spanish-speaking population by lee et al. it consists of 50 items and is based on multiple choice questions. a score between 0 and 37 suggests that the respondent has inadequate hl. after the protocolized translation process (translation/backtranslation), the final version of the croatian translation of sahlsa-50 was produced, after we introduced several changes that do not represent a direct translation of the spanish items. most of the changes were made because of a cultural difference in common use or in the meaning of the term. conclusion: sahlsa-50 was translated and adapted into croatian using the standard translation/back-translation procedure. the croatian version will now be available as an important part of epidemiological and clinical studies, as a useful tool for evaluating the levels of hl in the croatian-speaking population. validation of the translated croatian version in practice would be the next step. (berlančić t, kuharić m, janković d, milić j, badak k, zibar l. croatian translation and linguistic adaptation of the short assessment of health literacy for spanish adults (sahlsa-50). seemedj 2020; 4(2); 121-128) seemedj 2020, vol 4, no. 2 croatian translation of sahlsa-50 122 southeastern european medical journal, 2020; 4(2) introduction health literacy (hl) is a combination of skills, including the ability to perform basic reading and numerical tasks required to function in a healthcare environment (1). the definition used by the institute of medicine states that it is the degree to which individuals have the capacity to obtain, process and understand basic health information and services needed to make appropriate health decisions (2). hl has become an important and fruitful area of research, mostly because of the relationship of limited hl with patients’ knowledge, health behaviors, health outcomes and medical costs (3). today, there are multiple different assessment tools for assessing hl (4, 5). short assessment of health literacy for spanish adults (sahlsa-50) is an hl assessment tool containing 50 items designed to assess spanish-speaking adults’ ability to read and understand common medical terms (6). sahlsa-50 was originally developed and validated in spanish. it is an easy tool for researching hl in the general population (6). it has been validated for a spanish-speaking country – chile, and it was recognized as a useful tool for assessing hl in the adult chilean population. its use and incorporation into local research can be especially recommended in areas of education and health promotion (7). while the interest in hl was until recently mainly concentrated in the united states and canada, it has become more internationalized over the past decade (8). although the european union produced less than a third of the global research on hl between 1991 and 2005 (9, 10), the importance of the issue has been increasingly recognized in european health policies. as a case in point, hl is explicitly mentioned as an area of priority action in the european commission’s health strategy 2008-2013 (11). we chose sahlsa-50 as appropriate for the croatian language because of its structure, which is intended for a language that is written phonetically (spanish), unlike the english language. to the authors’ knowledge, there are no similar hl questionnaires in the croatian language to date. material and methods sahlsa-50 sahlsa-50 was developed for the spanishspeaking population by lee et al. (6), based on the rapid estimates of adult literacy in medicine (realm), known as the most easily administered tool for assessing hl in english. sahlsa-50 consists of 50 items and is based on multiple choice questions (specifically three choices). each item consists of a stem medical term and for each term, a respondent chooses between the two offered, potentially related terms, of which only one is right. the right term is called a key term and is in some way related to the stem medical term (synonym or descriptor). the other term is a distractor, which has nothing in common with the stem medical term (or at least in comparison with the key term). the third choice is “i do not know”, which is an option for avoiding guessing the answer. for each correct related term chosen, the respondent is given a point. a score between 0 and 37 suggests that the respondent has inadequate hl (5). the authors of the original questionnaire approved the translation into croatian language. procedure our translation was modeled after francisco et al. (12) and steps taken followed those described by wild et al. (13); they are shown in a flowchart (figure 1). the first step included the first forward translation from spanish into croatian by two of the authors of this paper (two students of medicine with passive knowledge of the spanish language) and one spanish speaking physician, all croats, with the help of a professor of the spanish language (a native speaker of croatian). in the second step, corrections were made by another professor of the spanish language (a native speaker of croatian). back-translation was done by a fluent bilingual native speaker of spanish living in croatia, who is also a spanish language teacher by profession. finally, the translation team made the harmonization, including the modification for croatian language. seemedj 2020, vol 4, no. 2 croatian translation of sahlsa-50 123 southeastern european medical journal, 2020; 4(2) the final version of the croatian-language version of sahlsa-50 was the result results table 1 shows sahlsa-50 versions during the translation process, from the original version to the final translated version in croatian. the final version of the croatian sahlsa-50 is available in supplement 1. the majority of the items was translated directly and without modification. several items underwent modification during the translation process. most of the changes were done due to cultural or linguistic differences or divergence in meaning. for example, in item 10, “vigilance” (“budnost”) in croatian is better associated with “coffee” (“kava”) than with the original “energy” (“energija”). in item 17, “small egg” (“jajašce”) has been replaced by ovulation (“ovulacija”), because it was less confusing, since in croatian an “egg” colloquially also means a “testicle”. another change was done because the authors wanted to achieve concordance with more common croatian expressions in terms of the word category that is typically used. thus, the adjective in item 22, “happy” (“sretan”), was replaced with “happiness” (“sreća”), which is a noun, and in item 23, the words “to lose”, which are a verb in croatian (“izgubiti”), replaced the word “loss”, which is a noun in croatian. regarding miscarriage, it is more common to say that someone lost the baby (“izgubili bebu” in croatian) and not that they experienced a “loss” (“izgubiti” vs “gubitak” in the record). another difference was introduced in item 6, where we changed “meals”, a plural noun (“obroci”), into a singular noun – “meal” (“jelo”), which is more common and less confusing ( “obroci” has an additional different meaning besides “meals”). through empirical medical knowledge it was concluded that in item 20, the word “irritation” (“iritacija”) in croatian usually relates to “inflammation” (“upala”) and not “pain” (“bol”), as was the case in the original spanish version. the final change was made with the word “convulsions” in item 49, which does not have a singular meaning in croatian, while the word “epilepsija”, meaning “epilepsy” (related to “convulsions”), is more common and does have a recognizable related croatian description in the word “padavica” (the term for a grand mal seizure characterized by falling down – as “pad” in “padavica”). the procedure of translation and back-translation went smoothly, without major differences between the attitudes, proposals or final solutions of the members of the translation team. the translation team agreed on the final version without lingering objections. discussion we made the croatian version of sahlsa-50 in order to enable hl research in croatia. the translation was made using standard validated translation procedures (12-14). since the spanish questionnaire was found to be more suitable than the english one, it was quite easy to translate the items. croatian and spanish have a simple way of pronouncing written text, where only one sound corresponds to each letter, and vice versa. thus, being literate in these two languages (spanish and croatian) is supposed to be comparably difficult (or easy), unlike english, where each word is pronounced in a certain way, depending on many complicated rules and exceptions. however, we had to make several changes of certain spanish words, since we wanted the questionnaire to be more appropriate for croats in terms of recognizable content and category of words used. similar changes in wording or scoring are commonly done to ensure cultural adaptation (15). we hope that the next step – the validation of the croatian version of sahlsa-50 in practice – will confirm the adequacy of our choice. the next step after this report is validation of the translated questionnaire in the croatian-speaking population. . seemedj 2020, vol 4, no. 2 croatian translation of sahlsa-50 124 southeastern european medical journal, 2020; 4(2) spanish original forward translation corrections back translation 1. próstata glándula circulación prostata žlijezda cirkulacija prostata žlijezda cirkulacija la próstata la glándula la circulación 2. empleo trabajo educación zaposlenje posao obrazovanj e zaposlenje posao obrazova nje el empleo el negocio la educación 3. menstrual mensual diario menstrual an mjesečno dnevno menstruacijs ko mjesečno dnevno menstrual mensual diario 4. gripe sano enfermo gripa zdrav bolestan gripa zdrav bolestan la influenza saludable enfermo 5. avisar medir decir upozoriti mjeriti reći upozoriti mjeriti reći advertir medir decir 6. comidas cena paseo obroci večera šetnja jelo večera šetnja la comida la cena el paseo 7. alcoholismo adicción recreo alkoholiza m ovisnost rekreacija, odmor alkoholizam ovisnost rekreacija el alcoholismo la adicción la recreación 8. grasa naranja manteca mast, masnoća naranča maslac, mast masnoća naranča maslac la grasa la naranja la mantequill a 9. asma respirar piel astma disati koža astma disati koža la asma respirar la piel 10. cafeína energía agua kofein energija voda kofein budnost voda la cafeína la vigilancia agua 11. osteoporosis hueso músculo osteoporo za kost mišić osteoporoza kost mišić osteoporosis el hueso el músculo 12. depresión apetito sentimient os depresija tek osjećaji depresija tek osjećaji la depresión el apetito los sentimient os 13. estreñimient o bloqueado suelto opstipacija zatvor, blokiran rastresit, nekomplet an konstipacija zatvor proljev el estreñimiento el estreñimient o la diarrea 14. embarazo parto niñez trudnoća porođaj djetinjstvo trudnoća porođaj djetinjstvo el embarazo el parto la infancia 15. incesto familia vecinos incest obitelj susjedi incest obitelj susjed el incesto la familia el vecino 16. pastilla tableta galleta pilula tableta dvopek, kolačić pilula tableta kolačić la píldora la tableta el pastelito 17. testículo óvulo esperma testis jajašce sperma testis ovulacija sperma el testículo la ovulación el semen 18. rectal regadera inodoro rektalan kanta za polijevanje wc, dezodoran s rektalno tuš wc rectal la ducha el inodoro 19. ojo oír ver oko čuti vidjeti oko sluh vid el ojo el oído la vista seemedj 2020, vol 4, no. 2 croatian translation of sahlsa-50 125 southeastern european medical journal, 2020; 4(2) 20. irritación rígido adolorido iritacija ukočen bolan iritacija ukočeno bolno la irritación tenso doliente 21. abnormal diferente similar abnormaln o drukčije slično abnormalno drukčije slično abnormal diferente similar 22. estrés preocupació n feliz stres zabrinutos t sretan stres zabrinutos t sreća el estrés la preocupació n la felicidad 23. aborto espontáneo pérdida matrimoni o spontani pobačaj gubitak brak spontani pobačaj izgubiti brak el aborto espontáneo perder el matrimonio 24. ictericia amarillo blanco ikterus, žutica žut bijel ikterus žuto bijelo la ictericia amarillo blanco 25. papanicolao u prueba vacuna papanicola u test kozice papanicolau test cjepivo papanicolaou la prueba la vacuna 26. impétigo pelo piel osip dlaka koža osip dlaka koža la erupción el cabello la piel 27. indicado instrucción decisión indiciran, označen uputa odluka naznaka uputa odluka la indicación instrucciones la decisión 28. ataque herida sano napad ozljeda zdrav napad ozlijeđen zdrav el ataque lesionado sano 29. menopausia señoras niñas menopauza gospođe djeca menopauza gospođe djevojke la menopausia señoras chicas 30. apéndice rascar dolor slijepo crijevo grepsti bol slijepo crijevo ogrebotina bol el apéndice el rasguño el dolor 31. comportamie nto pensamient o conducta ponašanje misao vladanje ponašanje misao vladanje el comportamie nto el pensamiento la conducta 32. nutrición saludable gaseosa prehrana zdrav gazirano piće prehrana zdravlje gazirano piće la nutrición la salud las bebidas carbonatad as 33. diabetes azúcar sal dijabetes šećer sol dijabetes šećer sol la diabetes el azúcar el sal 34. sífilis anticoncepti vo condón sifilis kontracept ivi kondom sifilis kontracept ivi kondom la sífilis anticoncepti vos el condón 35. inflamatorio hinchazón sudor upaljen oteklina znoj upala oteklina znoj la inflamación el hinchazón el sudor 36. hemorroides venas corazón hemoroidi vene srce hemoroidi vene srce las hemorroides las venas el corazón 37. herpes aire sexo herpes zrak seks, spol herpes zrak seks herpes el aire el sexo seemedj 2020, vol 4, no. 2 croatian translation of sahlsa-50 126 southeastern european medical journal, 2020; 4(2) 38. alérgico resistencia reacción alergičan otpornost reakcija alergija otpornost reakcija la alergia la resistencia la reacción 39. riñon orina fiebre bubreg urin groznica bubreg mokraća vrućica el riñon la orina la fiebre 40. calorías alimentos vitaminas kalorije hrana vitamini kalorije hrana vitamini las calorías la comida las vitaminas 41. medicamento instrumento tratamient o lijek instrument terapija lijek instrument terapija la medicina el instrumento la terapia 42. anemia sangre nervio anemija krv živac anemija krv živac la anemia la sangre el nervio 43. intestinos digestión sudor crijeva probava znoj crijeva probava znoj los intestinos la digestión el sudor 44. potasio mineral proteína kalij mineral protein kalij mineral protein el potasio el mineral la proteína 45. colitis intestino vejiga kolitis crijevo mokraćni mjehur kolitis crijeva mjehur el colitis los intestinos la vejiga 46. obesidad peso altura pretilost težina visina pretilost težina visina la obesidad el peso la altura 47. hepatitis pulmón hígado hepatitis pluća jetra hepatitis pluća jetra la hepatitis los pulmones el hígado 48. vesícula biliar arteria órgano žučni mjehur arterija organ žučni mjehur arterija organ la vesícula biliar la arteria el órgano 49. convulsione s mareado tranquilo grčevi, konvulzije bolestan od morske bolesti miran epilepsija padavica smirenost la epilepsia la epilepsia la calma 50. artritis estómago articulació n artritis želudac zglob artritis želudac zglob la artritis el estómago la articulación seemedj 2020, vol 4, no. 2 croatian translation of sahlsa-50 127 southeastern european medical journal, 2020; 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4(2) 14. milić j, škrlec i, milić vranješ i, matić m, sertić d, heffer m. the croatian translation of the horne and östberg morningness-eveningness questionnaire with a brief review of circadian typology. southeastern european medical journal. 2018; 2(1):1-11. 15. adan a, almirall h. horne & östberg morningness-eveningness questionnaire: a reduced scale. personality and individual differences. 1991; 12 (3): 241-253. i author contribution. acquisition of data: berlančić t, kuharić m, janković d, milić j, badak k, zibar l administrative, technical or logistic support: berlančić t, kuharić m, janković d, milić j, badak k, zibar l analysis and interpretation of data: berlančić t, kuharić m, janković d, milić j, badak k, zibar l conception and design: berlančić t, kuharić m, janković d, milić j, badak k, zibar l critical revision of the article for important intellectual content: berlančić t, kuharić m, janković d, milić j, badak k, zibar l drafting of the article: berlančić t, kuharić m, janković d, zibar l final approval of the article: berlančić t, kuharić m, janković d, milić j, badak k, zibar l guarantor of the study: berlančić t, zibar l provision of study materials or patients: berlančić t, badak k, zibar l seemedj 2020, vol 4, no. 2 total antioxidant status in cerebral vasoreactivity of copd 48 southeastern european medical journal, 2020; 4(2) original article the role of total antioxidant status in cerebral vasoreactivity of chronic obstructive pulmonary disease patients 1 marina hlavati *1,2, svetlana tomić 2, krunoslav buljan 2, silva butković-soldo 2 1 department for diagnostic and therapeutical procedures-neurology unit, general hospital našice, croatia 2 faculty of medicine osijek, josip juraj strossmayer university of osijek, croatia *corresponding author: marina hlavati, marinahlavati@yahoo.com received: mar 14, 2020; revised version accepted: oct 23, 2020; published: nov 12, 2020 keywords: chronic obstructive pulmonary disease, antioxidants, transcranial doppler ultrasonography, breathholding, cerebral perfusion abstract introduction: chronic obstructive pulmonary disease (copd) is associated with an oxidantantioxidant imbalance. copd patients have impaired cerebral vasoreactivity (cvr). impaired cvr could be correlated with total antioxidant status (tas) in plasma. aim: to determine the role of systemic tas in cvr of copd patients. material and methods: in this cross-sectional observational study, we included 120 participants (the mean age of 67±7.9, 87 males), 90 copd patients categorized according to the severity of airway obstruction in mild, moderate, severe/very severe and 30 ageand sex-matched controls. we analyzed baseline mean flow velocities (mfv) of the middle cerebral artery (mca) and the basilar artery (ba), the mean breath-holding index (bhim) of those arteries (bhimmca and bhimba) by transcranial doppler ultrasound and tas in plasma. the level of significance was set to α=0.05. results: a significant negative correlation between tas and bhimba (rho=−0.445, p=0.01) was found only in the mild copd group. in copd groups, baseline mfv ba is in a significant positive correlation with bhimmca and bhimba (rho=0.336, p=0.001 and rho=0.647, p<0.001). according to the severity of airway obstruction in copd groups, a significant positive correlation between baseline mfv ba and bhimba was found: in mild (rho=0.731, p<0.001), moderate (rho=0.574, p=0.001) and severe/very severe (rho=0.398, p=0.03). conclusion: systemic tas was not correlated with cvr in all copd groups. however, perfusion in the ba of copd groups was in a significant positive correlation with the anterior and posterior cvr. the analysis of perfusion in the basal cerebral arteries should be part of a future study of cvr in copd patients. (hlavati m, tomić s, buljan k, butković-soldo s. the role of total antioxidant status in cerebral vasoreactivity of chronic obstructive pulmonary disease patients. seemedj 2020; 4(2); 48-61) seemedj 2020, vol 4, no. 2 total antioxidant status in cerebral vasoreactivity of copd 49 southeastern european medical journal, 2020; 4(2) introduction the coexistence of chronic obstructive pulmonary disease (copd) and vascular disease is known, but the pathophysiology of this association has not been fully elucidated. the regulation of the cerebral blood flow (cbf) could be altered by some extrapulmonary consequences, such as arterial blood gas levels, acid-base imbalance, oxidative burden, endothelial dysfunction and autonomic disorders (1,2). the strongest regulator of the cbf is the arterial partial pressure of carbon dioxide (paco2). large changes in the flow could result even from its small fluctuations. the change in the cbf for a given increase in arterial carbon dioxide (co2) is greater than the change in the cbf observed for the same magnitude reduction in arterial co2 (3). copd patients have high oxidative stress associated with the severity of airway obstruction (4). oxidative stress occurs when the resident antioxidants are insufficient or fail to upregulate sufficiently to neutralize an increased oxidant burden (5). in copd, increased oxidative stress causes pulmonary inflammation. there is experimental and clinical evidence that pro-inflammatory mediators from the lungs overflow into the systemic circulation and cause the alteration of blood vessel structure. the vascular modelling and arterial stiffness lead to endothelial dysfunction. those dysfunctional cells reduce the bioavailability of the vasoactive substance they secrete, such as nitric oxide (no), which otherwise causes relaxation of smooth muscle cells lining in arterioles (6). the sensitivity of cerebral blood vessels to changes in paco2 is termed cerebral vasoreactivity (cvr), where arterial hypercapnia induces vessel dilatation and causes an increase in cerebral perfusion (7). damaged endothelial function is associated with impaired co2 reactivity, causing impairment of cvr. therefore, cvr could be a surrogate of cerebrovascular endothelial function (8). copd patients have impaired cvr in the anterior and posterior cerebral circulation and the impairment increases with the airway obstruction severity (9). a study that analyzed moderate smoking-related copd postmenopausal women showed altered cerebrovascular responses in the anterior cerebral circulation to hypercapnia. they exhibited increased oxidative stress, suggesting that it may be significant in the dysfunction of cvr observed during hypercapnic challenge in copd (10). different methods defining total antioxidant status (tas) of different biological samples have been developed and some of them can determine tas in the plasma of copd patients (11). the aim of this study was to determine the role of systemic tas in cvr of copd patients. material and methods study subjects a cross-sectional observational study was conducted at the general hospital between march and august 2018. one hundred twenty participants were included in the study – 90 copd patients (mild, moderate and severe/very severe group, each consisting of 30 participants) and 30 healthy volunteers with a mean age 67 ± 7.9, from which 87 males (student’s t test, p = 0.27). in terms of gender, there were no significant differences between the proportion of men and women in copd patients and the control group (74.4 vs. 66.7 and 25.6 vs. 33.3% respectively; p = 0.41; fisher's exact test). the baseline characteristics of copd patients and controls were summarized in table 1. the exclusion criteria were as follows: previous cerebrovascular disease, stenosis of internal carotid arteries (>50%), inadequate transtemporal acoustic window/s, atrial fibrillation, active malignity, exacerbation of copd in previous two months, specific therapies (nonsteroidal anti-inflammatory and antioxidant therapy, vitamins a, c and/or e supplementation). copd patients with arterial hypertension, hyperlipidemia, ischemic heart disease and diabetes mellitus that represent vascular disease risk factors were included if they were under good therapy control. seemedj 2020, vol 4, no. 2 total antioxidant status in cerebral vasoreactivity of copd 50 southeastern european medical journal, 2020; 4(2) table 1. baseline characteristics, lung functions, laboratory values and pack-years in copd patients and controls median (interquartile range) difference 95% confidence interval p* controls copd age (years) 64 (4) 67 (9) 1.8‡ -5.1 do 1.5 0.27 fev1/fvc ratio [median (interquartile range)] 0.80 (0.75 – 0.83) 0.61 (0.46 – 0.68) 0.20‡ 0.15 do 0.25 <0.001† fev1 (%) 106 (12.7) 65 (25.5) 40.7 33.6 do 47.7 <0.001 bmi (kg/m2) 27.2 (3.2) 28.7 (6.7) 1.5 -4.03 do 0.97 0.23 body temperature (°c) 35.3 (5.7) 35.9 (3.3) 0.64 -2.3 do 1.04 0.45 systolic blood pressure (mmhg) 130 (13) 135 (16) 5,2 -11.6 do 1.2 0.13 diastolic blood pressure (mmhg) 80 (5) 81 (6) 0,8 -3.5 do 1.8 0.53 hemoglobin (g/l) 137 (13) 143 (14) 5,7 -11.6 do 0.08 0.05 crp (mg/l) [median (interquartile range)] 2.2 (0.97 – 3.15) 2.6 (1.4 – 6.2) 0.6‡ -0.1 do 1.6 0.08† pack-years [median (interquartile range)] 41 (17.3 48) 43 (33 55) 9‡ -6.8 do 30 0.27† *student t test; †mann-whitney u test; ‡hodges-lehmann median difference fev1/fvc – forced expiratory volume in one second / forced vital capacity; fev1 − forced expiratory volume in one second; bmi – body mass index; crp – c-reactive protein; pack-years = number of cigarettes per day x number of smoking years / 20 study design the participants were divided into four groups. the first three groups were copd patients, divided according to spirometry findings and the global initiative for chronic obstructive lung disease (gold) classification of airflow obstruction severity based on forced expiratory volume in one second (fev1) as mild; moderate; and severe to very severe (12). according to gold, copd was defined by postbronchodilator fev1 to forced vital capacity (fvc) ratio (fev1/fvc) of <0.70, confirming a persistent airflow obstruction (13). the fourth group were controls − healthy volunteers (fev1/fvc >70% and fev1 >80% of the predicted value). anthropometric measurements, comprising height and weight, were determined. body mass index (bmi) was calculated and expressed as weight (kg) / height (m2). pack-years were calculated as the number of cigarettes per day x the number of years smoked /20 (14). the participants were asked to abstain from caffeine, alcohol and intense physical activity at least 12 hours prior to participation in the study. for all participants, the exclusion of significant internal carotid stenosis was performed by the method of color and power doppler flow imaging with a linear probe (vf 10-5 linear ultrasound transducer, siemens acuson x300, germany). for all participants, a specialized technician performed spirometry (spiroscoutᴿ ganshorn, germany) according to the american thoracic society/european respiratory society standardization (15). baseline cerebral perfusion and cvr were determined by transcranial seemedj 2020, vol 4, no. 2 total antioxidant status in cerebral vasoreactivity of copd 51 southeastern european medical journal, 2020; 4(2) doppler (tcd) ultrasound (digilite™, rimed, industrial park raanana, israel). the study was approved by the ethics committee of general hospital našice (no. 01497/3-2017) and by the ethics committee of the faculty of medicine osijek, josip juraj strossmayer university of osijek, croatia (no. 2158-61-07-17-209). all data were anonymized and the study was conducted in accordance with the amended declaration of helsinki. informed consent was obtained from all individual participants included in the study. all participants signed an informed consent form before entering the study. biochemical analysis venous blood was taken for a biochemical analysis. we analyzed the complete blood count (sysmax xn-1000 sa-01, sysmax europe gmbh), c-reactive protein (crp) and tas (beckman coulter dxc 700 au analyzer, usa). the plasma antioxidant status was measured using a commercially available tas kit (reagents randox laboratories ltd, united kingdom). the serum separated from blood was frozen at -20 ºc. the samples were collected over a period of 14 days and analyzed in one day by using a colorimetric assay. the procedure was repeated until the target number of participants was reached. the reference range was set at 1.30 – 1.77 and the results were expressed as mmol/l (16). other blood samples were analyzed immediately after being taken. tcd monitoring tcd monitoring of the middle cerebral arteries (mcas) that represent the anterior cerebral circulation was performed by previously described protocol (9,17,18). the breath-holding test was used as a hypercapnic stimulus. using an original headband device (rimed, industrial park raanana, israel), we secured both 2-mhz doppler probes over the trans-temporal windows. an optimal insonation position during monitoring was maintained and escaped movement artifacts. baseline mean flow velocity (mfvbaseline) was defined as a continuous mean velocity over 30 seconds during the resting period in supine position and normal breathing of room air. maximum mfv (mfvmax) was defined as the last 3 seconds of breathholding after normal inspiration. the minimum breath-holding time was set at 15 seconds and the maximum at 30 seconds. the test was repeated two times, with a resting period of 2 minutes. breath-holding index (bhi) was calculated as the difference in the increase of mfv (cm/s) occurring during breath-holding divided by the time (seconds) for which the participant held breath ([mfvmax -mfvbaseline] / breath-holding time). we were calculating bhi of each test on the right and left side and then the mean bhi (bhim) for each participant. the testing of the basilar artery (ba) that represents the posterior cerebral circulation was performed in a seated position, using the same handheld suboccipital insonation probe. testing was repeated twice to calculate bhim of the ba. among the croatian population, normal bhi values range from 1.03 to 1.65 (17). statistical analysis the sample size was calculated to be 120, effect size 0.35, level of significance 0.05 and power 0.9 (g*power software, version 3.1.9.2, by franz faul, university kiel, germany). local ethics committees approved the study. the category data are represented by absolute and relative frequencies. differences between categorical variables were tested by the χ2 test, and, if necessary, by the fisher exact test. the normality of the distribution of numeric variables was tested by the shapiro-wilk test. numerical data are described by the arithmetic mean and standard deviation in case of a normal distribution, and by the median and the limits of the interquartile range in cases where the distribution is not normal. the means of the numerical variables of interest were evaluated using a 95% confidence range. the student's ttest and mann-whitney's u test (with hodgeslehmann median difference) were used to test the differences in the numerical variables between two independent groups of subjects, depending on the normality of the distribution. the kruskal-wallis (post-hoc conover) test was seemedj 2020, vol 4, no. 2 total antioxidant status in cerebral vasoreactivity of copd 52 southeastern european medical journal, 2020; 4(2) used to test for differences in the numerical variables between three or more independent groups. the correlation of the numerical variables was evaluated by the spearman correlation coefficient ρ (rho). the differences in the numerical variables before and after testing were tested with the wilcoxon test (19). all p values were two-sided. the level of significance was set at alpha=0.05. medcalc statistical software version 18.11.3 (medcalc software bvba, ostend, belgium; https://www.medcalc.org; 2019) and spss (ibm corp. released 2015. ibm spss statistics for windows, version 23.0. armonk, ny: ibm corp.) were used for all analyses. results the baseline characteristics of copd patients according to the severity of airflow obstruction were summarized in table 2. table 2. baseline characteristics, lung functions, laboratory values and pack-years in copd groups median (interquartile range) p* mild moderate severe/very severe total age (years) 66 (59 – 70) 68 (62 – 72) 71 (65 – 78) 68 (62 – 74) 0.01 †fev1/fvc ratio [median (interquartile range)] 0.68 (0.67 – 0.69) 0.645 (0.57 – 0.68) 0.41 (0.34 – 0.48) 0.68 (0.67 – 0.69) <0.001 † fev1 (%) 93.5 (87.8 – 103.5) 64 (56 – 69.3) 37 (31 – 42.8) 64 (42 88) <0.001 bmi (kg/m2) 29.4 (26.9 – 31.7) 31.7 (25.9 35) 24 (19.9 – 29.2) 28.6 (24.1 33) <0.001 body temperature (0c) 36.2 (36 – 36.5) 36.1 (36 – 36.3) 36.3 (36 – 36.5) 36.2 (36 – 36.4) 0.26 systolic blood pressure (mmhg) 140 (130 150) 132.5 (120 146) 130 (120 – 142.5) 132.5 (120 150) 0.21 diastolic blood pressure (mmhg) 80 (80 90) 80 (80 85) 78 (70 80) 80 (80 80) 0.05 hemoglobin (g/l) 136 (130.5 – 150.3) 147.5 (135 154) 144 (131.75 158) 144 (133 -151) 0.44 crp (mg/l) [median (interquartile range)] 2.6 (1.3 – 4.6) 2.5 (1.6 – 6.2) 2.95 (1.3 – 7.73) 2.6 (1.4 – 6.2) 0.69 pack-years 40.5 (31.8 – 52.5) 43 (27.6 51) 49 (41.5 – 73.88) 43 (33.5 – 54.4) 0.15 *kruskal-wallis test (post-hoc conover); †on level p < 0.05 significant differences between mild vs. moderate, mild vs. severe/very severe, moderate vs. severe/very severe; ‡on level p < 0.05 significant differences between mild vs. severe/very severe, moderate vs. severe/very severe fev1/fvc – forced expiratory volume in one second / forced vital capacity; fev1 − forced expiratory volume in one second; bmi – body mass index; crp – c-reactive protein; pack-years = number of cigarettes per day x number of smoking years / 20 arterial hypertension was significantly less observed in the severe/very severe copd group (χ2 test, p = 0.003) and there were no differences in the presence of hyperlipidemia, ischemic heart disease and diabetes mellitus among the copd groups (data not presented). significant differences were found in baseline mfv of mca. in copd groups, higher velocity was on the left, and in controls on the right mca. seemedj 2020, vol 4, no. 2 total antioxidant status in cerebral vasoreactivity of copd 53 southeastern european medical journal, 2020; 4(2) baseline mfv of mca and ba and also bhimmca and bhimba were significantly lower in copd patients than in controls. tas was significantly higher in copd than controls (table 3). table 3. baseline mfv, cerebral vasoreactivity and tas in copd patients and controls median (interquartile range) p* controls p* copd p* mfv mca right (cm/s) 37 (29.8 – 44.3) <0.001 30 (23 35) <0.001 0.004 mfv mca left (cm/s) 34.5 (30 – 40.5) 31 (23 – 39.25) 0.01 mfv ba (cm/s) 29,5 (26 – 30.3) 24.5 (17 – 30.3) 0.003 bhimmca 1.24 (1.16 – 1.39) 0.8 (0.7 – 0.9) <0.001 bhimba 1.07 (1.02 – 1.15) 0.7 (0.6 – 0.9) <0.001 tas (mmol/l) 1.6 (1.5 – 1.7) 1.7 (1.5 – 1.8) 0.03 *mann-whitney u test; †wilcoxon test mfv – mean flow velocity; tas – total antioxidant status; mca – middle cerebral artery; ba – basilar artery; bhim – breath-holding index mean in copd groups, based on greater severity of the disease, there were significant reductions in baseline mfv of mca and ba and we found significant impairment of bhimmca and bhimba (table 4). table 4. baseline mfv, cerebral vasoreactivity and tas in copd groups median (interquartile range) p* mild p§ moderate p§ severe/very severe p§ mfv mca right (cm/s) 32 (29.8 39) 0.78 30.5 (18 – 39.25) 0.53 26 (19 – 32.5) 0.91 0.02† mfv mca left (cm/s) 32.5 (25.8 41) 31.5 (23 – 37.75) 24.5 (17.8 – 33.3) 0.04† mfv ba (cm/s) 28 (20.8 – 32.3) 22.5 (17 – 30.3) 19 (14.5 – 29.3) 0.04† bhimacm 0.94 (0.89 – 1.06) 0.8 (0.7 – 0.9) 0.7 (0.6 – 0.7) <0.001‡ bhimab 0.83 (0.72 – 1.03) 0.7 (0.6 – 0.9) 0.6 (0.4 – 0.7) <0.001‡ tas (mmol/l) 1.6 (1.52 – 1.78) 1.7 (1.6 – 1.8) 1.7 (1.6 – 1.8) 0.52 *kruskal-wallis test (post-hoc conover); †on level p < 0.05 significant differences between mild vs. severe/very severe; ‡on level p < 0.05 significant differences between mild vs. moderate, mild vs. severe/very severe, moderate vs. severe/very severe; §wilcoxon test mfv – mean flow velocity; tas – total antioxidant status; mca – middle cerebral artery; ba – basilar artery; bhim – breath-holding index mean seemedj 2020, vol 4, no. 2 total antioxidant status in cerebral vasoreactivity of copd 54 southeastern european medical journal, 2020; 4(2) using the spearman's rank correlation coefficient, a significant negative correlation was found between age and bhimba in controls. in copd groups, we found a significant negative correlation between age and bhimmca and bhimba. in the copd group, baseline mfv of ba is in a significant positive correlation with bhimmca and bhimba (table 5). table 5. correlation between age and baseline mfv ba with bhim mca and bhim ba in copd patients and controls spearman's rank correlation coefficient rho (p value) age (year) mfv ba (cm/s) controls bhimmca -0.089 (0.64) 0.051 (0.79) bhimba -0.376 (0.04) 0.124 (0.51) copd bhimmca -0.375 (< 0.001) 0.336 (0.001) bhimba -0.376 (< 0.001) 0.647 (<0.001) mfv – mean flow velocity; bhim – breath-holding index mean; mca – middle cerebral artery; ba – basilar artery tas and bhimba significantly negatively correlated only in copd groups, but not in the control group (figure 1). figure 1. the correlation between total antioxidant status (tas) and mean breath-holding index of the basilar artery (bhimba) in copd patients and controls using the spearman's rank correlation coefficient rho, we found a significant negative correlation between tas and bhimba (rho= −0.327, p=0.002) in copd patients and no significant correlation in controls. seemedj 2020, vol 4, no. 2 total antioxidant status in cerebral vasoreactivity of copd 55 southeastern european medical journal, 2020; 4(2) figure 2. the correlation between total antioxidant status (tas) and mean breath-holding index of the basilar artery (bhimba) according to copd severity using the spearman's rank correlation coefficient rho, we found a significant negative correlation between tas and bhim ba (rho= −0.445, p=0.01) in the mild copd groups and no significant correlations in other copd groups. there were no significant correlations between tas and bhimmca in copd groups, nor between tas and bhimmca in the control group (data not presented). using the spearman's rank correlation coefficient rho, based on the severity of copd and age, a significant negative correlation was found between age and bhimba (rho= −0.398, p=0.03) in the moderate group. in the severe/very severe copd group, a significant negative correlation was found between age and bhimmca (rho= −0.451, p= 0.01), but there were no significant correlations in other copd groups (data not presented). in terms of the severity of airway obstruction, we found a significant negative correlation between tas and bhimba only in the mild copd (figure 2). there was no significant correlation between tas and bhimmca in any copd group (data not presented). in all copd groups, a significant positive correlation waws found between baseline mfv ba and bhimba (figure 3), but there was no correlation between mfv ba and bhimmca (data not presented). seemedj 2020, vol 4, no. 2 total antioxidant status in cerebral vasoreactivity of copd 56 southeastern european medical journal, 2020; 4(2) figure 3. the correlation between baseline mean flow velocity of the basilar artery (mfv of ba) and mean breath-holding index of the basilar artery (bhim ba) based on copd severity using the spearman's rank correlation coefficient rho, we found a significant positive correlation between baseline mfv ba and bhimba in all copd groups: in mild (rho=0.731, p<0.001), moderate (rho=0.574, p=0.001) and severe/very severe copd (rho=0.398, p=0.03). discussion the aim of the study was to analyze the relationship between cvr and tas in the plasma of copd patients and controls. in copd patients, tas was in a significant negative correlation with cvr in the posterior cerebral circulation, but in terms of the severity of airflow obstruction, we found a negative correlation between cvr and tas in the posterior cerebral circulation only in the mild copd group. there was no significant correlation between cvr and tas in the anterior cerebral circulation. the results from this study do not allow conclusions that impaired cvr is associated with systemic changes in the antioxidative status of copd patients. a significantly higher tas values were found in copd patients than in controls, which contrasts the previously published data. the reason for that could be multifactorial, but determining the mechanisms for was in the scope of our recently published manuscript, where we demonstrated that tas is a predictor of copd (20). in terms of gender, there were more men participating in the study than women, but they were equally represented in the copd groups and in the control group. all women in the study have reached menopause. in that period, oxidative stress was reported to increase because of the decreasing levels of estrogen, which has beneficial vasoactive and antioxidative effects (21). a study of postmenopausal women with moderate copd seemedj 2020, vol 4, no. 2 total antioxidant status in cerebral vasoreactivity of copd 57 southeastern european medical journal, 2020; 4(2) related to smoking analyzed oxidative stress and cvr in the anterior cerebral circulation. the study showed an impaired cerebrovascular response to hypercapnia, significantly higher levels of oxidative stress, a higher level of glutation peroxidase that represents antioxidant enzyme activity and a higher ratio between oxidative stress and antioxidant activity than in controls. there was no significant correlation between the markers of oxidative stress/antioxidants and cvr measured by tcd. the only correlation was found between higher catalase activity, which represents antioxidant enzyme activity associated with higher ventilatory response sensitivity. in the context of higher oxidative stress caused by the decline of estrogen levels in menopause, it was concluded that that could be a possible explanation of the observed differences in cerebrovascular sensitivity to hypercapnia between copd women and controls (10). hypoxemia is part of clinical features of copd patients. a recent study analyzed how lifetime exposure to hypoxia (3,600 m above sea-level, la paz, bolivia) affects oxidative stress, cvr and cognitive function. chronic mountain sickness is a maladaptation syndrome experienced at high altitudes, characterized by severe hypoxemia. the syndrome is related to a higher risk of stroke, migraine, increased morbidity and mortality. it has been confirmed that individuals with such syndrome have exaggerated oxidative stress and a corresponding decrease in vascular no bioavailability. oxidative stress was associated with an impaired cerebrovascular function. the authors of this study concluded that oxidative stress induced by hypoxemia is associated with blunted cerebral perfusion, impaired cvr to hypercapnia, accelerated cognitive decline and depression (18). in our study, copd patients had significantly impaired cvr. impaired cvr, that is impaired co2 vasoreactivity as a surrogate of endothelial dysfunction, results in a decline of no (8). a recent study on an animal model showed that an increase in the bioavailability of no through prostaglandin pathways causes vasodilatation; therefore, it increases oxygenation and produces higher tas. that way, the antioxidant – tas eliminates free radicals from biological systems (22). this conclusion is based on a previous study suggesting that as the tas value increases, free radicals decrease, which protects macromolecules from damage (23). a recent study analyzed ageand apolipoprotein e (apoe) gene-specific hemodynamic changes using the blood-oxygen-level-dependent (bold) functional magnetic resonance imaging (mri) method. the breath-holding method was used for the examination of cvr and visual stimulation tasks to evoke functional hyperemia. after a three-day intake of nitrate as a no source, there was no effect on cvr or functional hyperemia, but cvr significantly decreased with age and was dependent on the genotype of participants. that way, the response of vascular functions on breath-holding could be dependent on the apoe-genotype and independent of no (24). such an analysis far exceeds our technical capabilities, but it would certainly be useful to include a genetic analysis in the endothelial dysfunction analysis of copd patients, in order to successfully manage therapeutic decisions, evaluate the response to target interventions and provide prognostic information. in our study, there were differences between the baseline right and left anterior cerebral perfusion in copd patients and controls. determining the mechanism(s) involved in those differences was not the goal of this study. our opinion is that the differences observed were not significant for the ultimate results of the study. in copd, the baseline anterior and posterior cerebral perfusion was significantly lower than in controls. based on greater severity of copd, a significant decline of perfusion was recorded in the anterior and posterior cerebral circulation. the baseline posterior cerebral perfusion was in a significant positive correlation with cvr of the anterior and posterior cerebral circulation in copd, which was not found in controls. in terms of the severity of airway obstruction, all copd groups had a significant positive correlation between the baseline posterior cerebral seemedj 2020, vol 4, no. 2 total antioxidant status in cerebral vasoreactivity of copd 58 southeastern european medical journal, 2020; 4(2) perfusion and cvr in the posterior cerebral circulation. decreased cerebral perfusion means reduced cerebral blood flow velocity. tcd perfusion analysis assumes that there is no change in the width of the artery lumen and that the blood flow is laminar. despite that interpretation constraint of tcd, in the study, in cases when lower cerebral perfusion was recorded, that decrease correlated with an impaired ability of the cerebral arteries to respond to vasodilator stimuli, such as hypercapnia (18,25,26). in our study, a significant negative correlation was found between age and cvr in the posterior cerebral circulation only in the moderate copd group and between age and cvr in the anterior cerebral circulation in the severe/very severe copd group. therefore, the results do not allow a final conclusion on the correlation between the age of copd patients and cvr in our sample. in the study that used functional tcd and hypercapnic stimuli as a marker of cvr, the cerebral perfusion changes were determined as a response to the language and arithmetic task in healthy young, healthy old and old subjects with the risk factors for atherosclerosis. arterial hypertension was the most frequent risk factor and the breath-holding method was used as a hypercapnic stimulus. the cerebral perfusion changes were significantly lower during the cognitive task and hypercapnia in the group of old subjects with vascular risk factors compared to the healthy young and old subjects. these results showed that the presence of cardioand cerebrovascular risk factors significantly reduces the ability of cerebral vessels to react to vasoactive stimuli. aging alone, without such risk factors, could not be responsible for hemodynamic changes caused by neuronal activation (27). in the study that used the pulsed arterial spin labelling perfusion mri and bold methods, the regional cerebral blood flow and cvr were assessed in young and elderly participants. age-related decreases in the baseline cbf and cvr were observed in the cerebral cortex, which may be related to the vulnerability to neurological disorders in aging (28). detection of early dysfunction of the cerebral vascular system in older people with the risk of developing cognitive impairment ensure the possibility of vascular-specific procedures which help maintain cognitive function through a lifetime (29). the tcd ultrasound technique does not allow clear spatial resolution of cvr. the main limitation of the study was that tas was the only biomarker that we performed because of technical and financial reasons. in comparison with clinical characteristics and individual biomarker, the combination of copd markers increases the prognostic value for proper outcome monitoring (30,31). we did not analyze any of the oxidants, so the increased oxidative burden in our population is not clearly proven. on the other side, the choice of peripheral blood among other biological samples is more appropriate because of the non-invasiveness of blood sampling and its property of easily allowing repeated measurements (11). the main limitations of tcd, and therefore of our study, are its dependency on the operator, the presence of adequate insonation windows and the possibility of recording inaccurate mean flow velocities due to wrong insonation angles (32). the strength of tcd is that the estimation of cerebral blood flow velocity, that is cerebral perfusion and cvr by that method, using co2 as a hypercapnic stimulus, has acceptable levels of reproducibility. it is an appropriate method for determining the effect of hypercapnia on cerebral haemodynamics (33). the correlations between cvr and systemic levels of the antioxidant marker of tas in the plasma have not been demonstrated in all copd groups. however, based on the decrease in perfusion in the anterior and posterior cerebral circulation detected in copd patients compared to controls, there is a possible correlation between cvr and the initial perfusion in the basal cerebral arteries. according to previous studies, there are correlations between impaired cvr and the risk of stroke, progressive global vascular damage, an increased risk of mortality, especially seemedj 2020, vol 4, no. 2 total antioxidant status in cerebral vasoreactivity of copd 59 southeastern european medical journal, 2020; 4(2) cardiovascular mortality, cognitive decline and dementia (34–38). early detection of copd patients at risk of these comorbidities presents an opportunity for early therapeutic interventions aimed at reducing such risks. conclusion in our study, a negative correlation between cvr and the plasma level of tas was found only in the posterior cerebral circulation in the mild copd group. the results do not allow the conclusion that impaired cvr is associated with systemic changes in the antioxidative status of copd patients. however, we found that decreased perfusion in the basilar artery of copd patients was in a significant positive correlation with cvr of the anterior and posterior cerebral circulation, which was not observed in controls. the analysis of perfusion in the basal cerebral arteries should be part of a future study of cvr in copd patients. acknowledgement. we gratefully acknowledge the time and effort of our research participants. disclosure funding. no specific funding was received for this study. competing interests. none to declare. ireferences 1. 1. van de ven mjt, colier wnjm, van der sluijs mc, kersten btp, oeseburg b, folgering h. ventilatory and cerebrovascular responses in normocapnic and hypercapnic copd patients. eur respir j. 2001; 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00:1–13. 36. portegies mlp, de bruijn rfag, hofman a, koudstaal pj, ikram ma. cerebral vasomotor reactivity and risk of mortality: the rotterdam study. stroke. 2014; 45(1):42–7. 37. wolters fj, de bruijn rfag, hofman a, koudstaal pj, arfan ikram m. cerebral vasoreactivity, apolipoprotein e, and the risk of dementia: a population-based study. arterioscler thromb vasc biol. 2016; 36(1):204– 10. 38. van der thiel m, rodriguez c, de ville d van, giannakopoulos p, haller s. regional cerebral perfusion and cerebrovascular reactivity in elderly controls with subtle cognitive deficits. front aging neurosci. 2019; 10:1–12. ii i author contribution. acquisition of data: hlavati m, tomić s, buljan k, butković-soldo s administrative, technical or logistic support: hlavati m, tomić s, buljan k, butković-soldo s analysis and interpretation of data: hlavati m, tomić s, buljan k, butković-soldo s conception and design: hlavati m, tomić s, buljan k, butković-soldo s critical revision of the article for important intellectual content: hlavati m, tomić s, buljan k, butković-soldo s drafting of the article: hlavati m, tomić s, buljan k, butković-soldo s final approval of the article: hlavati m, tomić s, buljan k, butković-soldo s provision of study materials or patients: hlavati m, buljan k statistical expertise: hlavati m, tomić s, buljan k, butković-soldo s iiabbreviations. copd − chronic obstructive pulmonary disease; cbf − cerebral blood flow; paco2 − arterial partial pressure of carbon dioxide; co2 − carbon dioxide; no − nitric oxide; cvr − cerebral vasoreactivity; tas − total antioxidant status; gold − global initiative for chronic obstructive lung disease; fev1 − forced expiratory volume in one second; fev1/fvc − forced expiratory volume in one second fev1 to forced vital capacity fvc ratio; bmi − body mass index; tcd − transcranial doppler ultrasound; crp − c-reactive protein; mca − middle cerebral artery; mfv − mean flow velocity; bhim − breath-holding index mean; ba − basilar artery; apoe − apolipoprotein e; bold mri − blood-oxygen-leveldependent magnetic resonance imaging method. seemedj 2021, vol 5, no. 1 slide in centric on students 176 southeastern european medical journal, 2021; 5(1) original article slide in centric on a random sample of students of the school of medicine in split 1 ivana strikić đula 1, nikolina lešić 2*, davor seifert 3 1 health centre šibenik, croatia 2 dental office seifert d.o.o., faculty of dental medicine and health, j. j. strossmayer university, osijek, croatia; school of medicine, university of split, croatia. 3 faculty of dental medicine and health, j. j. strossmayer university, osijek, croatia; school of medicine, university of split, croatia; dental office seifert d.o.o., corresponding author: nikolina lešić, nikolina.lesic@gmail.com received: aug 18 2020; revised version accepted: mar 20, 2021; published: apr 28, 2021 keywords: dental occlusion, centric; temporomandibular joint dysfunction syndrome abstract introduction: a slide in centric is defined as a slide from centric relation to maximum intercuspation. understanding contact between natural teeth is important for longevity of the stomatognathic system, diagnosis and therapy planning. the aim of this study was to determine the difference in the length of slide in centric in population according to dental status, sex and previous orthodontic therapy. materials and methods: the study was conducted on a sample of 33 students at the university of split, school of medicine (dental study). results: slide values do not follow normal or gaussian distribution according to the kolmogorov– smirnov test (p<0.05). for that reason, they were represented by the median as a measure of central tendency. the arithmetic mean of a slide in centric is 0.95 mm ± 0.47 mm. a slide in centric was not present in only 10% of the subjects. a slide between 0.5 mm and 1.5 mm to maximum intercuspation was present in 90% of the examinees. there was no statistically significant difference in the length of slide between the subjects who had all teeth and those who had missing teeth 1-4 (z= 0.507; p= 0.612). there was no significant difference in the length of slide between women and men (z= 0, p=1). there was no significant difference in the length of slide between the patients who underwent orthodontic therapy and those who did not (z=0.253; p=0.800). conclusion: there is some controversy about slide in centric and its etiological role in the development of temporomandibular disorders. slide in centric is very significant because it indicates occlusal instability and can eventually lead to temporomandibular dysfunction, which do not have to be of the same aetiology.. (strikić đula i, lešić n, seifert d. i slide in centric on a random sample of students of the school of medicine in split. seemedj 2021; 5(1); 176-182) seemedj 2021, vol 5, no. 1 slide in centric on students 177 southeastern european medical journal, 2021; 5(1) introduction understanding the contact of teeth in certain positions and during the movement of the mandible is important for longevity of the stomatognathic system, diagnosis, planning prosthodontic treatment, and treatment of dysfunctions (1). occlusion principles are gnathological or of a “freedom in centric” type. in the gnathological type of occlusion, maximum intercuspation is equal to centric relation. therefore, there are no initial contacts during the closing movement around the kinematic axis. this type of occlusion is called centric occlusion (2). a group of gnathological or organic occlusion ensures that during laterotrusion, contacts occur only on cuspids, while any other contact or slide on any other tooth represents interference (3). numerous authors, among whom lauritzen (4), define cuspid guided occlusion as physiological and thus desirable. cuspid guided occlusion is generally found in young population. the other type of occlusion is ”freedom in centric” (4, 5, 6). posselt was the first author to describe the concept of ”freedom in centric” (7). the freedom in centric concept allows a slight initial contact of the antagonists during the closing movement of the mandible, around the kinematic axis, and a slide to the maximum contact between the lower and upper teeth. this slide is considered normal and physiological only if it occurs in the sagittal direction. a slide in centric is defined as movement from the initial contact of the antagonistic teeth in centric occlusion to maximum intercuspation. the slide is approximately 0.5-1 mm. it is acceptable when it occurs in the anterior direction (8). mann and pankey use the term “long centric” to describe the case where there is an anterior slide between the retruded contact position and habitual occlusion in a length of approximately 1 mm (9). freedom in centric defines the possibility of movement from the initial centric contacts to maximum intercuspation in all orthogonal planes: the horizontal, frontal, and sagittal plane. over time, the attitude about physiological relations in the temporomandibular joint has been changing. centric relation and its definition have been evolving for years. there are at least 25 definitions of centric relation (10). initially, it was the posterior superior position of the condyle in relation to fossa articularis to an anterior superior position. the currently recognised centric relation definition indicates the maxillo-mandibular relation in which the condyle articulates with the thinnest avascular part of the articular disc with the disc-condyle complex in the anterior-superior position against the inclined plane of the articular eminences (11). therefore, it is also the most distal unstrained physiological relation of the mandible against the maxilla, from which lateral movements are possible. despite differences between definitions, centric relation is a repeatable position and it is used as a reference position in prosthetic treatment. only in 10% of the population does maximum intercuspation coincide with centric relation, which represents a mutually protected occlusion or gnathological occlusion. regarding the rest of the population, there is a difference between the initial contact in centric relation (retruded cuspal position) and maximum intercuspation. this slide is approximately 0.5-1.5 mm. changing occlusal surface due to prosthetic rehabilitation, a prosthodontic appliance or a dental filling can cause a premature contact during the closing movement in central relation and consequently to the loss of equilibrium or pathological occlusion (12). slide in centric of 2 mm is one of the most important occlusal parameters pointing to joint pathology (13, 14) and relates to mandibular instability (15). some studies have confirmed the influence of slide in centric (in a length of over 2 mm) on joint pathology (16). as far as such slide exists between the position of centric relation and maximal intercuspation, diagnoses given in clinical practice can very often reveal pain in the lateral pterygoid muscle. this muscle pain disorder represents temporomandibular dysfunction and can be easily misunderstood for intracapsular temporomandibular disorders. the aim of this study was to determine if slide in centric occurs in young healthy population seemedj 2021, vol 5, no. 1 slide in centric on students 178 southeastern european medical journal, 2021; 5(1) without any signs of temporomandibular dysfunction according to dental status, sex and previous orthodontic treatment (with or without therapy). materials and methods a cross-sectional study was conducted as a clinical examination on each subject. it included a random sample consisting of student volunteers of the university of split, school of medicine (dental study). all students were informed about the study and 33 students signed an informed consent. of the total sample tested, 25 persons were students of dental medicine and 8 were students of medicine. the youngest subject was 20 years old and the oldest subject was 24 years old. in terms of sex, there were 13 men and 20 women participating in the study. the mean age of female subjects was 22.4+/-1.2 and the mean age of male subjects was 22.6+/-1.1 years. the mean age of the entire sample was 22.4+/-1.2 years. from the total sample tested, 21 subjects underwent previous orthodontic treatment, whereas 12 participants did not. during the clinical examination, decayed, missing, and filled permanent teeth (the dmft index) was defined for each student sample. slide in centric of every participant was measured using a wax bite record. the participants were positioned in a dental chair in an upright position, with the head resting on a headrest. a warmed, trimmed and softened wax plate was adjusted to the maxillary dental arch. mouth closing in centric relation was achieved using the dawson bimanual guiding technique to the point of initial contact between the mandibular teeth and the wax bite plate. closing under guidance continued until the wax plate was bitten through and the initial contact of the antagonistic teeth was made. after registering the initial contact in centric relation, participants bit the wax plate to the point of maximum intercuspation. after removing the wax plate from the mouth, the length of slide was measured using a caliper. the statistical analyses used included the kolmogorov– smirnov test and descriptive analyses. results in this clinical examination, dental status was recorded. eleven male subjects and 14 female subjects had all teeth, representing 79% of the sample. one male subject and two female subjects were missing one tooth, representing 9% of the sample. one male subject and one female subject (3% of the sample) had two missing teeth. one female subject (3% of the sample) had three missing teeth and two female subjects (6% of the sample) had four missing teeth. veneer or other dental restorations were not observed in any of the participants. orthodontic therapy was administered to eight male subjects and 13 female subjects or 64% of the sample. the mean value of slide in centric in the entire sample was 1 mm (ranging from 0 mm to 1.5 mm). regarding the male subjects, the values of slide in centric measured ranged from 0 to 1.5 mm, with a mean value of 1 mm. the same values were obtained upon examination of 20 female subjects. the slide values do not follow normal or gaussian distribution according to the kolmogorov–smirnov test (p<0.05). for that reason, they were represented by the median as a measure of central tendency (minmax). arithmetic mean of slide in centric was 0.95 mm ± 0.47 mm. based on the dental status of the subjects with all teeth, slide in centric of 1 mm was observed in 25 subjects, ranging from 0 to 1.5 mm, using the mann–whitney u test 0.612. slide in centric of 1 mm (0-1.5 mm) was equally observed in both genders using the mann–whitney u test value of 1.0. participants who underwent orthodontic therapy had slightly higher values of slide in centric – 0-1.5 mm – compared to the participants who did not undergo orthodontic therapy, whose values ranged from 0 to 1.4 mm, mann–whitney u test 0.800. table 1 shows the subjects according to variables in relation to slide in centric (no centric, positive side). of 25 participants who had all teeth, 21 of them had slide in centric with an initial contact during closure in centric relation, with an equal distribution between male and female subjects. a higher percentage of existing slide in centric was observed in the participants who underwent orthodontic therapy. seemedj 2021, vol 5, no. 1 slide in centric on students 179 southeastern european medical journal, 2021; 5(1) table 1. subjects according to variables in relation to slide in centric (no slide, positive slide) slide in centric 0 (no slide) slide dental status have all teeth 4 21 missing 1-4 teeth 2 6 sex women 4 16 men 1 12 orthodontic therapy no 1 12 yes 4 16 no statistically significant difference in the length of slide in centric between the examined subjects who have all teeth and those who had missing teeth 1-4 (z= 0.507; p= 0.612) was observed in this study. furthermore, no significant difference in the value of slide in centric between women and men (z= 0; p = 1) was observed in this study. a statistically significant difference in the value of slide in centric between the subjects who underwent and the subjects who did not undergo orthodontic therapy (z = 0.253; p = 0.800) was not observed. table 3 shows the number of subjects according to the variables studied in relation to slide in centric (no slide, slide in centric present). slide in centric was not observed in only five subjects. discussion and conclusion there is some controversy about slide in centric and its etiological role in the development of temporomandibular disorders. in 1918, harris observed slide in centric of 1 mm or less. only in 10 % of the population does centric relation coincide with central occlusion, but in 90 % of the population, a slide from retruded contact position to maximum intercuspation occurs. mandible slide (there is a slide) in an amount of 0.5 mm-1.5 mm. results of this study comply with the data from the literature because slide in centric was not observed in only five subjects (33). ramfjord and ash (17) and froemden (18) assume that the freedom in centric occlusion increases proportionally with age, based on the degree of tubercle abrasion. results of this survey do not support ramfjord’s, ash’s and froemden’s opinion. this study was carried out seemedj 2021, vol 5, no. 1 slide in centric on students 180 southeastern european medical journal, 2021; 5(1) on a compact and young age group and it indicates a large percentage of slide in centric occurrence. some researchers state that slide in centric over 2 mm has a significant etiological impact on temporomandibular dysfunctions (pvalue of 0.008) (15, 19). using a sample of 749 patients, nilner showed that temporomandibular dysfunction correlates with slide in centric (20). gnathologists confirm that malocclusion contributes to the pathology of the temporomandibular joint by selective grinding after orthodontic treatment (21). in this study, a statistically significant difference in slide in centric between the subjects who have all teeth and the subjects with missing 1-4 teeth (z = 0.507; p = 0.612) was not found, as shown in table 1. those results point out the fact that functional adaptation persists by entrenched neuromuscular adaptation after a partial loss of the teeth. although a stable relation between the mandible and maxilla in the maximum habitual intercuspation existed before the loss of teeth, it persisted during partial loss of teeth. initial contact of the remaining teeth during the closing movement initiates the same movements as a jaw with teeth. in this study sample, there was no significant difference in the length of slide in centric between the subjects who underwent orthodontic therapy and those who did not (z = 0.253; p = 0.800). this was also assumed by haralur, who confirmed, in his study on a sample of 36 patients (who underwent orthodontic therapy) and a control group (who did not undergo orthodontic therapy), that the length of slide in centric and orthodontic therapy were not correlated (22). there is no significant difference in the length of slide between women and men (z= 0, p=1). it is obvious that sexual dimorphism was not observed in our study. in some studies, there is no correlation between slide in centric and temporomandibular disorders (23). slide in centric is highly significant because it indicates a premature contact during the closure of the jaw and possible occlusal instability, which can eventually lead to temporomandibular dysfunction, which do not have to be of the same aetiology. in contrast, huber and hall stated in their study that slide does not affect the temporomandibular joint (24). there is no common opinion about slide in centric potential pathology because the central nervous system could diminish potentially damaging forces through neuromuscular control and the compensatory mechanism. the measurements and applied methodology are basic, useful in everyday practice and dental offices lacking sophisticated equipment. accuracy is not on a high level, but can provide useful information for practitioners. such a study should be repeated on a more comprehensive sample in order to identify the differences between each group more easily. by using a greater sample, the result will be more valuable. however, this investigation included only student volunteers, who participated in the study during one semester. therefore, the sample is relatively small. nevertheless, the data obtained in this study can provide useful information for practitioners. in everyday work, the difference between the closure of the jaw in centric relation and slide of 1 mm can produce harmful forces on teeth and tmjs as well as cause muscle fatigue and pain. using a simple and quick method, the practitioner can obtain necessary information regarding the type of occlusion. by doing so, it is possible to avoid interreference during reconstructive procedures. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. seemedj 2021, vol 5, no. 1 slide in centric on students 181 southeastern european medical journal, 2021; 5(1) references 1. anderson jr, myers ge. nature of contacts in centric occlusion in 32 adults. j dental res. 1971; 50:7. doi: 10.1177/00220345710500013201. 2. hodge lc, mahan pe jr. a study of mandibular movement from centric occlusion to maximum intercuspation. j prosthet dent. 1967; 18(1):19–30. doi: 10.1016/0022-3913(67)90107-2. 3. schmidt diemel k. okklusionkorrektur in gnathologischer sicht. teil 2. dtsch zahnarztl z 1970; 26:1006-14. 4. lauritzen ag. function, prime object of restorative dentistry, a definite procedure to obtain it. j am dental assoc 1951; 42:532-4. 5. santos jd jr. gnathologie. prinzipien und konzepte. koln: deutsche aerzte-verlag 1988; 129-44. 6. schmidt diemel k. okklusionkorrektur in gnathologischer sicht. dtsch zahnarztl z 1970; 26: 918-24. 7. posselt u. studies in the mobility of human mandibule. acta odontol scand 1952; 10: (suppl 10) 19-160. 8. hodge lc, mahan pe jr. a study of mandibular movement from centric occlusion to maximum intercuspation. j prosthet dent. 1967; 18(1):19–30. doi: 10.1016/0022-3913(67)90107-2. 9. mann aw, pankey ld. oral rehabilitation. part i. useof the pm instrument in treatment planning and restoring the lower posterior teeth. j prosth dent 1960; 10: 135-50. 10. rinchuse dj, kandasamy s. centric relation: a historical and contemporary orthodontic perspective. j am dent assoc. 2006; 137:494–501. 11. the glossary of prosthodontics terms. j prosthet dent. 2005; 94:10-92. 12. ehrlich j, taicher s. intercuspal contacts of the natural dentition in centric occlusion. j prosthet dent. 1981; 45:419–421. doi: 10.1016/0022-3913(81)90104-9. 13. pullinger ag, seligman da. quantification and validation of predictive values of occlusal variables in temporomandibular disorders using a multifactorial analysis. j prosthet dent. 2000; 83(1):66–75. 14. kirveskari p, jamsa t. health risk from occlusal interferences in females. eur j orthod. 2009; 31(5):490–5. 15. seligman da, pullinger ag. analysis of occlusal variables, dental attrition, and age for distinguishing healthy controls from female patients with intracapsular temporomandibular disorders. j prosthet dent. 2000; 83(1):76–82. 16. okeson jp. occlusion and functional disorders of the masticatory system. dent clin north am. 1995; 39(2):285–300. 17. ramfjord s, ash m orthodox. occlusion iii, phila­delphia london toronto mexico city rio de janeiro sydney tokio: wb sounders, 1983. 18. frömder b. untersuchungen über zusammen­hänge zwischen der zahnführung bei laterotrusion und der bialalge. dtsch zahnartzl z 1989; 44:77-82. 19. satheesh b haralur. digital evaluation of functional occlusion parameters and their association with temporomandibular disorders. j clin diagn res. 2013; 7(8):1772–1775. published online 2013 jul 19. doi: 10.7860/jcdr/2013/5602.3307 20. nilner m. functional disturbances and diseases of the stomatognathic system. a crossectional study. j pedond. 1986; 10:211-238. 21. mcnamara ja jr, seligman da, okeson jp. occlusion, orthodontic treatment, and temporomandibular disorders: a review. journal of orofacial pain. 1995; 9(1):73-90. 22. haralur sb digital evaluation of functional occlusion parameters and their association with temporomandibular disorders. j clin diagn res. 2013; 7:1772-5. doi: 10.7860/jcdr/2013/5602.3307. epub 2013 jul 19 seemedj 2021, vol 5, no. 1 slide in centric on students 182 southeastern european medical journal, 2021; 5(1) 23. zonnenberg aj1, mulder j. the incidence of centric slides in healthy individuals and tmd patients. eur j prosthodont restor dent. 2013; 21:109-13. 24. huber ma1, hall eh. a comparison of the signs of temporomandibular joint dysfunction and occlusal discrepancies in a symptom-free population of men and women. oral surg oral med oral pathol. 1990; 70:180-3.. author contribution. acquisition of data: strikić đula i, lešić n, seifert d administrative, technical or logistic support: strikić đula i, lešić n, seifert d analysis and interpretation of data: strikić đula i, lešić n, seifert d conception and design: strikić đula i, lešić n, seifert d critical revision of the article for important intellectual content: strikić đula i, lešić n, seifert d drafting of the article: strikić đula i, lešić n, seifert d final approval of the article: strikić đula i, lešić n, seifert d seemedj 2021, vol 5, no. 1 duration of diabetes mellitus and progression of diabetic retinopathy 65 southeastern european medical journal, 2021; 5(1) original article association between diverse diabetic treatments and duration of diabetes mellitus according to progression of diabetic retinopathy: experience from a small regional hospital 1 ana bardak 1, stjepan kovacevic 2, bozidar kovacevic 3, zeljka vukovic arar 3, sandra sekelj 3, dinko nizic 4, zvonimir bosnic 5* 1 faculty of medicine osijek, josip juraj strossmayer university of osijek, osijek, croatia 2 department of internal medicine, general hospital “dr josip benčević”, slavonski brod, croatia 3 department of ophthalmology, general hospital “dr josip benčević”, slavonski brod, croatia 4 department of radiology and ultrasound diagnostics, special hospital for pulmonary diseases, zagreb, croatia 5 postgraduate interdisciplinary university study – molecular biosciences, josip juraj strossmayer university of osijek, croatia corresponding author: zvonimir bosnic, zbosnic191@gmail.com received: jan 9, 2021; revised version accepted: feb 1 2021; published: apr 28, 2021 keywords: diabetes complication, diabetic retinopathy, insulin-dependent diabetes, education abstract introduction: research objectives of present study were to examine sex and age-related specifics of diabetic retinopathy according to the therapy approach and duration of diabetes mellitus. the study also aimed to determine the association between the presence of diabetic retinopathy and diabetes duration as a prognostic factor of retinopathy progression in such patients. materials and methods: the study was designed as a retrospective study and included 289 patients with diabetic retinopathy, who were treated at the department of ophthalmology of the general hospital “dr. josip benčević” in slavonski brod during the period from 2019 to 2020. results: 176 patients were treated with oral antidiabetic drugs (oad), while 113 patients were insulindependent. the median age of patients treated with oad was 77 years. diabetic retinopathy was present in 35 (19.9%) patients, of whom 33 (18.8%) had non-proliferative diabetic retinopathy, while 2 patients (1.1%) had proliferative diabetic retinopathy. the median age of the insulin-dependent patients was 79 years. diabetic retinopathy was present in 54 patients (47.8%), non-proliferative diabetic retinopathy was diagnosed in 51 patients (45.1%), while proliferative diabetic retinopathy was diagnosed in only 3 (2.7%) patients. there was a significant difference between the presence of diabetic retinopathy and diabetes duration (p<0.001), as well as between the therapy approach and diabetes duration (⍺<0.001). conclusion: various hypotheses have been proposed to explain the worsening of diabetic retinopathy, and we assume that the therapy approach, duration of diabetes and hba1c have a significant role in retinopathy progression. hereby, we emphasize that, although there have been significant advances, there is still a pressing need for a better understanding of a new therapeutic modality, new tools for identifying high-risk patients and continued monitoring in order to intervene effectively before vision loss occurs. further research is needed to identify and implement the best practices to increase diabetic eye screening rates in the long term. (bardak a, kovacevic s, kovacevic b, vukovic arar z, sekelj s, nizic d, bosnic z. association between diverse diabetic treatments and duration of diabetes mellitus according to progression of diabetic retinopathy: experience from a small regional hospital. seemedj 2021; 5(1); 65-74) seemedj 2021, vol 5, no. 1 duration of diabetes mellitus and progression of diabetic retinopathy 66 southeastern european medical journal, 2021; 5(1) introduction diabetic retinopathy (dr) is one of the most frequent complications of diabetes mellitus and it remains a leading cause of vision loss globally. its aetiology and pathology have been extensively studied for half a century, but unfortunately, there are few therapeutic options and prevention of progression is still the ultimate goal. it affects an estimated 126.6 million people worldwide and it is expected to increase rapidly in the future (1). even though many studies, such as the diabetes control and complications trial (dcct) and the united kingdom prospective diabetes study (ukpds), have confirmed a strong relationship between chronic hyperglycaemia and the development and progression of diabetic retinopathy, there is a lack of understanding of the underlying mechanism leading to the development of microvascular damage (2, 3). according to the who, diabetic retinopathy is described as a major cause of 5% of vision loss in the developed world and its prevalence is expected to double by 2030. as stated by the american diabetes association, 21% of patients with diabetes mellitus have diabetic retinopathy at the moment of first diagnosis of diabetes, and more than 60% of patients develop dr within 20 years after the diagnosis (4). microvascular damage slowly accumulates in the retinal blood vessels, leading to retinal ischemia, higher retinal permeability, neovascularization and macular oedema, finally resulting in complete vision loss (5-7). the risk of development and progression of diabetic retinopathy is closely associated with the type and duration of diabetes, blood sugar levels, blood pressure levels, proteinuria and possibly hyperlipidaemia (8). recent studies suggest that apolipoproteins, inflammatory factors and genetic risk factors could also play a role in the development and progression of diabetic retinopathy (9). an ideal model of screening tools for diabetic retinopathy is based on an annual examination of visual acuity and the eye fundus in all diabetic patients. adults with type 2 diabetes should undergo an eye screening test at the time of diabetes diagnosis. annual eye exams are recommended, but if there is no evidence of diabetic retinopathy, eye screening every two years thereafter may be considered (10). family medicine physicians have adequate knowledge and awareness of diabetic eye screening guidelines. however, they encounter barriers in ensuring that patients undergo screening due to burdensome and complex tasks they are required to complete during the patient’s average 15-20-minute visit to the clinic, as well as due to a lack of access to the patients’ eye exam records. patients should undergo follow-ups by an experienced ophthalmologist using precise eye fundus imaging methods at least once a year. examination of the eye fundus completed with fluorescein angiography make a gold standard in retinopathy diagnosis and classification (11). research objectives of the present study were to examine sex and age-related specifics of diabetic retinopathy according to the therapy approach, duration of diabetes mellitus, as well as accompanying comorbidities. the study also aimed to determine the association between the presence of diabetic retinopathy, diabetes duration and hba1c as prognostic factors of retinopathy progression in such patients. materials and methods the retrospective study was conducted on 289 patients treated at the department of ophthalmology of the general hospital “dr. josip benčević” in slavonski brod, croatia during the period from 2019 to 2020. the data were collected from the medical records kept by the department of ophthalmology of the general hospital “dr. josip benčević”. the inclusion criteria were the presence of diabetes mellitus and biomicroscope presence of diabetic retinopathy. all procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 declaration of helsinki and its later amendments or comparable ethical standards. seemedj 2021, vol 5, no. 1 duration of diabetes mellitus and progression of diabetic retinopathy 67 southeastern european medical journal, 2021; 5(1) statistical methods categorical data were presented by absolute frequency and percentage, while numerical data were presented by the median, minimum, maximum and interquartile range. differences in nominal variables were tested by the fisher’s exact test, while differences in numeric variables were tested by the mann–whitney u test because of deviations from the normal distribution. all p-values were two-sided. the level of significance was set at α= 0.05. ibm spss statistics was used for the statistical analysis (ibm corp. released 2015. ibm spss statistics for macintosh, version 23.0. armonk, ny: ibm corp.). results this study was conducted on 289 patients, who were divided into two groups based on the therapy approach. the first group (n=176) was treated with oral antidiabetic drugs (oad), while the second group of patients (n=113) was insulindependent (table 1 and table 2). table 1. characteristics of patients (n = 176) treated with oral antidiabetic drugs frequency percentage gender male 72 40.9% female 104 59.1% smoking yes 5 2.8% no 171 97.2% alcohol consumption yes 2 1.1% no 174 98.9% hypertension yes 160 90.9% no 16 9.1% diabetic retinopathy yes 35 19.9% no 141 80.1% non-proliferative diabetic retinopathy yes 33 18.8% no 143 81.3% proliferative diabetic retinopathy yes 2 1.1% no 174 98.9% seemedj 2021, vol 5, no. 1 duration of diabetes mellitus and progression of diabetic retinopathy 68 southeastern european medical journal, 2021; 5(1) table 2. characteristics of insulin-dependent patients (n=113) frequency percentage gender male 55 48.7 % female 58 51.3 % smoking yes 9 8.0 % no 104 92.0 % alcohol consumption yes 2 1.8 % no 111 98.2 % hypertension yes 101 89.4 % no 12 10.6 % diabetic retinopathy yes 54 47.8 % no 59 52.2 % non-proliferative diabetic retinopathy yes 51 45.1 % no 62 54.9 % proliferative diabetic retinopathy yes 3 2.7 % no 110 97.3 % the median age of patients treated with oad was 77 years (interquartile range of 71-84), with a minimum age of 50 years and a maximum age of 95 years. the median age of the insulindependent patients was 79 years (interquartile range of 71-83), with a minimum age of 47 years and a maximum age of 96 years. out of 176 patients who received oad, 35 (19.9%) had diabetic retinopathy, while 141 (80.1%) did not. a total of 113 patients were insulin-dependent, of whom 54 (47.8%) had diabetic retinopathy and 59 (52.2%) did not (table 3). the oad group included 72 male patients (40.9%) and 104 female patients (59.1%). five patients (2.8%) were smokers and 171 patients (97.2%) were non-smokers. frequent alcohol consumption was reported by 2 patients (1.1%), while 174 patients (98.9%) did not consume alcohol frequently. hypertension was diagnosed in 160 patients (90.9%), while 16 patients (9.1%) did not have hypertension. diabetic retinopathy was present in 35 patients (19.9%), while 141 patients (80.1%) were not diagnosed with diabetic retinopathy. out of 176 patients, 33 patients (18.8%) had non-proliferative diabetic retinopathy, while 2 patients (1.1%) had proliferative diabetic retinopathy (table 1) seemedj 2021, vol 5, no. 1 duration of diabetes mellitus and progression of diabetic retinopathy 69 southeastern european medical journal, 2021; 5(1) table 3. characteristics of patients receiving oral antidiabetic drug therapy (oad) or insulin depending on the presence of diabetic retinopathy oad insulin total p• patients with diabetic retinopathy 35 (19.9) 54 (47.8) 89 (30.8) <0.001 patients without diabetic retinopathy 141 (80.1) 59 (52.2) 200 (69.2) <0.001 total 176 (100) 113 (100) 289 (100) fisher’s exact test the insulin-dependent group consisted of 55 men (48.7%) and 58 women (51.3%). out of 113 patients, 9 (8.0%) were smokers and 104 (92.0%) were non-smokers. frequent alcohol consumption was reported by 2 patients (1.8%). hypertension was diagnosed in 101 patients (89.4%), while 12 patients (10.6%) did not have hypertension. diabetic retinopathy was present in 54 patients (47.8%), while 59 patients (52.2%) were not diagnosed with diabetic retinopathy. non-proliferative diabetic retinopathy was diagnosed in 51 patients (45.1%), while the proliferative type was diagnosed in only 3 patients (2.7%). the groups differed based on diabetes duration. patients receiving oad had a median duration of diabetes of 7 years (interquartile range of 4-12 years), with a minimum duration of 6 months and a maximum duration of 35 years. a median duration of diabetes in the insulin-dependent patients was 12 years (interquartile range of 7-20 years), with a minimum duration of 3 months and a maximum duration of 55 years. patients diagnosed with diabetic retinopathy had a median duration of diabetes of 14 years (interquartile range of 8.5-20 years), with a minimum duration of 0 months and a maximum duration of 20 years. the patients without diabetic retinopathy had a median duration of diabetes of 7 years (interquartile range of 4-12 years), with a minimum duration of 6 months and a maximum duration of 55 years (figure 1). figure 1. difference in diabetes duration distribution between patients receiving oral antidiabetic drugs and insulin (a) and between patients with and without diabetic retinopathy (b). p < 0.001 both in figure 1a and figure 1b. seemedj 2021, vol 5, no. 1 duration of diabetes mellitus and progression of diabetic retinopathy 70 southeastern european medical journal, 2021; 5(1) patients diagnosed with diabetic retinopathy had a median duration of diabetes of 14 years (interquartile range of 8.5-20 years), with a minimum duration of 0 months and a maximum duration of 20 years. on the other hand, the patients without diabetic retinopathy had a median duration of diabetes of 7 years (interquartile range of 4-12 years), with a minimum duration of 6 months and a maximum duration of 55 years. hba1c concentrations were determined in the patients treated with oad and insulin. the patients treated with oad had a significantly lower median concentration of 6.3 mmol/l (interquartile range of 6.1-6.9), with a minimum concentration of 5.5 and a maximum concentration of 7.8 mmol/l. patients treated with insulin had a median concentration of 7.6 mmol/l (interquartile range of 6.6-8.9), with a minimum hba1c concentration of 5.5 and a maximum concentration of 12.3 mmol/l (figure 2). figure 2. difference in hba1c concentration between patients receiving oral antidiabetic drugs and insulin (a) and between patients with and without diabetic retinopathy (b). p < 0.001 both in figure 2a and figure 2b. discussion diabetic eye screening and treatment guidelines are part of the core curriculum for training eye care providers, but the current eye care provider workforce is insufficient to meet the increasing number of diabetic patients. according to epidemiological data, the number of diabetic patients is growing. these patients comprise a large share of eye care provider clinic time, but only one in 20 patients has vision-threatening diabetic eye disease. our study included 289 patients who suffered from type 2 dm. patients were divided into two groups according to the therapy approach. there were more patients who used oad. the majority of the patients using oad were females who suffered from arterial hypertension, did not consume alcohol and did not smoke. most of them had no signs of diabetic retinopathy and only two patients had confirmed proliferative diabetic retinopathy (table 1). in contrast to them, there was a similar number of male and female patients who were insulin-dependent. most of them suffered from arterial hypertension and half of them had confirmed dr. only three insulin-dependent patients had proliferative dr. the insulindependent patients had more cases of dr confirmed compared to patients who were taking oad (table 3). despite a relatively high prevalence of dr in our study, our results are close to the prevalence of dr reported in the seemedj 2021, vol 5, no. 1 duration of diabetes mellitus and progression of diabetic retinopathy 71 southeastern european medical journal, 2021; 5(1) region-specific information (europe) and worldwide (table 3) (12). some researchers have pointed out a higher prevalence of dr in patients treated with insulin, suggesting that insulin therapy may be associated with dr and dr severity when compared to the oral antidiabetic drug (oad) therapy (13). on the other hand, another study, conducted by gupta et al., has also shown a higher prevalence of dr among insulin users than in patients treated with oad (52.9% vs 16.3%), discussing how insulin therapy is often started later in the course of the disease, at a stage when glycaemic control is suboptimal for the subject. it was also argued that insulin is simply a marker of disease severity, rather than an independent risk factor for dr, suggesting that starting insulin therapy earlier in the course of the disease might be more beneficial in preventing the development of dr in the longer run (14). in the eurodiab study, mild forms of non-prolferative diabetic retinopathy (npdr) were recorded in 25.8%, moderate npdr in 9.8%, and pdr in 10.6% of insulin-treated patients. the study included 3250 insulin-treated diabetic patients from 13 european diabetes centres, with a mean diabetes duration of 14.7 years. the major factors for vision loss are patient age, diabetes duration, glycosylated haemoglobin and the grade of retinopathy (15, 16). dm duration is a predictor of diabetic retinopathy (17). patients with type 1 diabetes develop diabetic retinopathy within five years or less, and only occasionally later, i.e. 27% and 71-90% of patients with diabetes duration of 5-10 and >10 years, respectively. at 20-30 years of diabetes duration, the incidence of diabetic retinopathy increases to 95%. usually 30-50% of these patients develop proliferative diabetic retinopathy (pdr) (18). in our study, the patients receiving oad had a median duration of diabetes of 7 years (interquartile range of 4-12 years), while the median duration of diabetes in the insulin-dependent patients was 12 years (interquartile range of 7-20 years). the median age of the patients receiving oad was 77 years (iqr of 71-84), while the median age of the patients treated with insulin was 79 years (iqr of 71-83). there was a significant difference between the presence of diabetic retinopathy and diabetes duration (⍺<0.001) (figure 1a). thus, most of the patients were elderly persons with a comorbidity (e.g. arterial hypertension). even though the prevalence of dm is relatively high among elderly patients, the incidence of dr and pdr in our study is close to the results of studies conducted in other european countries (12). there are several factors which could explain these observations, some of which are a relatively high quality of life in the last 10 years, newly designed oad and combinations of oad, free physical examinations twice or thrice a year and morphological characteristics of the eye structure in elderly persons (posterior vitreous detachment), resulting in slower progression of dr than that observed (19). only a few participants in present study consumed alcohol, so alcohol can be excluded as a risk factor and is not directly associated with the presence or progression of diabetic retinopathy (table 1 and table 2). our results are similar to the multicentric study by lee cc et al., who investigated the association between alcohol consumption and diabetic retinopathy and deterioration of visual acuity in individuals with type 2 diabetes, concluding that alcohol consumption is associated with an increased risk of deterioration of visual acuity, but not with retinopathy in individuals with type 2 diabetes (20). the relationship between cigarette smoking and diabetic retinopathy was examined earlier and data suggest that there is no excess risk of retinopathy in smokers or ex-smokers when contrasted with those who have never smoked. our study produced similar results due to the fact that a small number of participants consumed cigarettes (table 1 and table 2) (21). landmark multi-centre, randomised controlled trials showed that early identification and proper treatment can prevent the risk of vision loss by 90%, but fewer than 50% of people with diabetes in the usa follow diabetic eye screening guidelines and even lower screening rates (1020%) have been described (22). once retinopathy is present, the duration of diabetes appears to be a less important factor than glycaemic control in forecasting progression from earlier to later stages of retinopathy (23). on the other hand, the seemedj 2021, vol 5, no. 1 duration of diabetes mellitus and progression of diabetic retinopathy 72 southeastern european medical journal, 2021; 5(1) link between hba1c levels and diabetic retinopathy is not conclusive because there are other variables that come into play. in our study, the patients treated with oral diabetic drug therapy had a significantly lower median concentration of hba1c, without the presence of diabetic retinopathy (figure 2a and b). maintaining control of glucose and blood pressure lowers the risk of retinopathy progression and patients should be aware of the importance of maintaining good levels of glycosylated haemoglobin and blood pressure. considering a higher prevalence of dm globally, family medicine doctors should improve additional educational programs in diabetic retinopathy screening because multiple workflow and systems-level barriers affect care providers and there is not enough time to follow all diagnostic features in everyday clinical practices (24). the study by olafsdottir e et al. discusses the benefits of regular screening for diabetes mellitus and diabetic eye disease as the gold standard in preventing diabetic blindness. according to that study, the loss of vision from diabetic retinopathy is uncommon if regular screening is provided and subsequent hospital costs are also lower. the same idea has been confirmed in the study by bandurskastankiewicz e et al., who have confirmed that the incidence of vision loss due to diabetes is significantly lower in the countries which have introduced programs for preventing retinopathy than in those countries which do not have such programs (25, 26). while current evidence indicates that the association between the glucagon-like peptide-1 receptor agonists (glp1ra), sodium-glucose cotransporter-2 (sglt-2) inhibitors and dipeptidyl peptidase-4 (dpp-4i) inhibitors and the risk of dr remains uncertain in patients with t2dm, future studies should focus on such types of drugs, especially on the combinations and prevalence of dr, pdr and npdr in large-scale, well-designed studies (27). once retinopathy is present, the duration of diabetes appears to be a less important factor than glycaemic control in forecasting progression from earlier to later stages of retinopathy. in our study, there is a lack of information about the type of oad and dr, so in future studies, we will pay more attention to the association of dr, new oad and combinations of therapy. in addition, opportunities exist in leveraging team-based care approaches, patient self-management programs and emerging telemedicine imaging technologies. conclusion our study has confirmed the results of previous studies, namely that the risk of development and progression of diabetic retinopathy is closely associated with the type and duration of diabetes and hba1c concentration. also, we emphasise that there is still a pressing need for a better understanding of a new therapeutic modality, new tools for identifying high-risk patients and continued monitoring in order to intervene effectively before vision loss occurs. further research is needed to identify and implement the best practices to increase diabetic eye screening rates in the long term. there is a lack of additional educational programs in primary health care of diabetic retinopathy screening and a lack of large-scale, well-designed studies of diabetic retinopathy occurrence associated with glucose-lowering drugs. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. zheng y, he m, congdon n. the worldwide epidemic of diabetic retinopathy. indian j ophthalmol. 2012; 60(5):428–31. doi:10.4103/0301-4738.100542. 2. white nh, cleary pa, dahms w, goldstein d, malone j, tamborlane wv. beneficial effects of intensive therapy of diabetes during adolescence: outcomes after the conclusion of the diabetes control and seemedj 2021, vol 5, no. 1 duration of diabetes mellitus and progression of diabetic retinopathy 73 southeastern european medical journal, 2021; 5(1) complications trial (dcct). j pediatr. 2001; 139(6):804–812. 3. tarr jm, ¸kaul k, chopra m, kohner em, chibber r. pathophysiology of diabetic retinopathy. isrn ophthalmol. 2013; 2013:343560. 4. ruta lm, magliano dj, lemesurier r, taylor hr, zimmet pz, shaw je. prevalence of diabetic retinopathy in type 2 diabetes in developing and developed countries. diabet med. 2013; 30:387– 398. 5. ahsan h. diabetic retinopathy – biomolecules and multiple pathophysiology. diabetes metab syndr clin res rev. 2015; 9(1):51– 4. 6. marcovecchio ml, lucantoni m, chiarelli f. role of chronic and acute hyperglycemia in the development of diabetes complications. diabetes technol ther. 2011; 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4(20):395–395. 14. gupta a, delhiwala k, raman rg, sharma t, srinivasan s, kulothungan v. failure to initiate early insulin therapy a risk factor for diabetic retinopathy in insulin users with type 2 diabetes mellitus: sankara nethralaya-diabetic retinopathy epidemiology and molecular genetics study (sn-dreams, report number 35). indian j ophthalmol. 2016; 64(6):440. 15. leske mc, wu sy, hennis a, nemesure b, schachat ap, hyman l, yang l; barbados eye study group. nine-year incidence of diabetic retinopathy in the barbados eye studies. arch ophthalmol. 2006; 124(2):250-5. 16. zoega gm, gunnarsdóttir t, björnsdóttir s, hreietharsson ab, viggósson g, stefánsson e. screening compliance and visual outcome in diabetes. acta ophthalmol scand. 2005; 83(6):687-90. 17. klein r, klein be, moss se, davis md, demets dl. the wisconsin epidemiologic study of diabetic retinopathy. iii. prevalence and risk of diabetic retinopathy when age at diagnosis is 30 or more years. arch ophthalmol. 1984; 102(4):527-532. doi:10.1001/archopht.1984.01040030405011. 18. yanko l, goldbourt u, michaelson ic, shapiro a, yaari s. prevalence and 15-year incidence of retinopathy and associated characteristics in middle-aged and elderly diabetic men. br j ophthalmol. 1983; 67(11):759765. doi:10.1136/bjo.67.11.759. 19. garancini p, moffitt p, valsania p, et al. prevalence of retinopathy in diabetic subjects from out-patient clinics in lombardy (italy), and associated risk factors. a multicenter epidemiologic study. diabetes res clin pract. 1989; 6:129–138. seemedj 2021, vol 5, no. 1 duration of diabetes mellitus and progression of diabetic retinopathy 74 southeastern european medical journal, 2021; 5(1) 20. lee cc, stolk rp, adler ai, et al. association between alcohol consumption and diabetic retinopathy and visual acuity-the adrem study. diabet med. 2010; 27(10):11301137. doi:10.1111/j.1464-5491.2010.03080.x 21. klein r, klein be, davis md. is cigarette smoking associated with diabetic retinopathy? am j epidemiol. 1983; 118(2): 228-238. doi:10.1093/oxfordjournals.aje.a113630. 22. lee pp, feldman zw, ostermann j, brown ds, sloan fa. longitudinal rates of annual eye examinations of persons with diabetes and chronic eye diseases. ophthalmology. 2003; 110(10):1952–9. doi:10.1016/s01616420(03)00817-0). 23. davis md, fisher mr, gangnon re, et al. risk factors for high-risk proliferative diabetic retinopathy and severe visual loss: early treatment diabetic retinopathy study report number 18. invest ophthalmol vis sci. 1998; 39(2):233-252 24. munoz b, o’leary m, fonseca-becker f, rosario e, burguess i, aguilar m, fickes c, wesk sk. knowledge of diabetic eye disease and vision care guidelines among hispanic individuals in baltimore with and without diabetes. arch ophthalmol. 2008; 126(7):968–74. doi:10.1001/archopht.126.7.968 ). 25. olafsdottir e, andersson dk, stefánsson e. visual acuity in a population with regular screening for type 2 diabetes mellitus and eye disease. acta ophthalmol scand. 2007; 85(1):4045. doi:10.1111/j.1600-0420.2006.00753.x 26. bandurska-stankiewicz e, wiatr d. programy prewencji utraty wzroku z powodu cukrzycy [programme preventing vision loss due to diabetes]. klin oczna. 2007; 109(7-9):359-362. 27. tang h, li g, zhao y, wang f, gower ew, shi l, wang t. comparisons of diabetic retinopathy events associated with glucose-lowering drugs in patients with type 2 diabetes mellitus: a network meta-analysis. diabetes obes metab. 2018; 20(5):1262-1279. doi: 10.1111/dom.13232. . author contribution. zvonimir bosnic and bozidar kovacevic were responsible for the conceptualisation and design of the study. stjepan kovacevic and dinko nizic performed the investigation and collected the data. in addition, they were responsible for data validation. ana bardak performed the statistical analysis. zeljka vukovic arar and bozidar kovacevic provided participants with the data. sandra sekelj and zeljka vukovic arar supervised the study. zvonimir bosnic and ana bardak wrote the manuscript. bozidar kovacevic and sandra sekelj reviewed and edited the manuscript. all authors have read and agreed on the published version of the manuscript seemedj 2020, vol 4, no. 2 sleep disorders in neurological diseases 35 southeastern european medical journal, 2020; 4(2) original article sleep disorders in cervical dystonia, parkinson’s disease and depression – what is the difference? 1 svetlana tomic *1,2, dunja degmecic 2,3, fabian gjoni 2, iva dumencic 2, snezana milanovic 1, tihana gilman kuric 1,2, zvonimir popovic 1,2, tea mirosevic zubonja 1,2 1 clinical department of neurology, university hospital centre osijek, croatia 2 faculty of medicine, josip juraj strossmayer university of osijek, croatia 3 clinical department of psychiatry, university hospital centre osijek, croatia *corresponding author: svetlana tomic, svetlana.tomic@vip.hr received: may 27, 2020; revised version accepted: oct 23, 2020; published: nov 12, 2020 keywords: sleep disorders; cervical dystonia; parkinson`s disease; depression; pittsburgh sleep quality index; epworth sleepiness scale abstract introduction: sleep disorders are among the most common non-motor symptoms in patients with cervical dystonia (cd), parkinson's disease (pd), and depression. the study aimed to assess the prevalence and characteristics of sleep disorders in patients with cervical dystonia compared to healthy controls, patients with parkinson's disease, and patients with depression. methods: in this cross-sectional study, we evaluated 122 patients (30 control patients, 30 with cervical dystonia, 32 with parkinson's disease, and 30 with depression). demographic data were collected. all of them, except for the depression group, were tested for depression and anxiety using the beck depression inventory (bdi) and beck anxiety inventory (bai). sleep disorders were evaluated using the pittsburgh sleep quality index (psqi) and epworth sleepiness scale (ess). statistical significance was defined at α=0.05. results: patients with cervical dystonia differed from the healthy control group in terms of psqi score and some subscales. the depression group differed in most psqi subscales when compared to the patients with parkinson's disease and cervical dystonia, while the latter two groups of patients differed only in the duration subscale. patients with parkinson's disease differed from other groups of patients only in one subscale daytime sleepiness. conclusion: cervical dystonia patients suffer from more sleep disturbances when compared to healthy controls. there are differences in the frequency and extent of sleep disturbances with less pronounced symptoms in patients with cervical dystonia and parkinson's disease, while patients with depression present the most pronounced symptoms. symptoms of depression and anxiety correlate with sleep disturbances in patients with parkinson's disease and cervical dystonia. patients with cervical dystonia do not experience daytime sleepiness problems. (tomic s, degmecic d, gjoni f, dumencic i, milanovic s, gilman kuric t, popovic z, mirosevic zubonja t. sleep disorders in cervical dystonia, parkinson’s disease and depression – what is the difference? seemedj 2020; 4(2); 35-47) seemedj 2020, vol 4, no. 2 sleep disorders in neurological diseases 36 southeastern european medical journal, 2020; 4(2) introduction circadian rhythm can be defined as changes in biological and behavioural conditions between the states of high and low activity during 24 hours. it is regulated by two paired nuclei located in the hypothalamus, called the suprachiasmatic nucleus (scn). the process starts with retinal light stimulation that generates a signal through the retinohypothalamic tract to the scn, which then generates a signal that stimulates the pineal gland. the process of stimulation involves a multi-synaptic link through the superior cervical ganglion, which releases noradrenaline during the night, inducing the activity of the serotonin-nacetyltransferase (snat) enzyme via cyclic adenosine monophosphate (camp). this stimulates the pineal gland to produce the hormone melatonin during the night and release it into the circulation to facilitate sleep [1]. the neurotransmitters that are important in the process of wakefulness are serotonin, noradrenaline, and acetylcholine, and they are released by the neurons located in the ventrolateral preoptic nucleus [2]. introduction to the rapid-eye-movement (rem) phase of sleep is characterized by a decrease in monoaminergic (serotonin, norepinephrine, and dopamine) tone with a parallel increase in cholinergic tone [3]. in non-rem sleep, glutamate signalling is important during thalamocortical slow oscillations [4]. the secretion of the hormone cortisol is decreased during normal deep sleep and hypercortisolaemia can induce insomnia [5]. sleep disorders could be classified into several types: delayed sleep phase syndrome (dsps), advanced sleep phase syndrome (asps), irregular sleep-wake pattern, and non-24-hour sleep-wake syndrome in blind and sighted persons. delayed sleep phase syndrome is a sleep disorder where the patient has problems falling asleep and waking up at conventional times, and the assumption is that the pathophysiological basis lies in delayed endogenous melatonin secretion [6]. advanced sleep phase syndrome is characterized by persistent early evening sleep onset and early morning awakening with no sleep-maintenance problems. it is an age-related problem, the pathophysiology of which is as a consequence of diminution in the output of the circadian pacemaker [7]. the syndrome of an irregular sleep-wake pattern is characterized by temporally disorganized and variable episodes of sleeping and waking behaviour. focal dystonia is characterized by sustained or intermittent muscle contraction causing abnormal, often repetitive movement, posture, or both. cervical dystonia (cd) is a focal dystonia type where neck muscles are involved [8]. the aetiology of cd is still unknown. recent articles suggest miscommunication between the basal ganglia and cerebellar loops [9]. in cd, besides typical motor symptoms, patients also experience non-motor symptoms, such as depression, anxiety, cognitive decline, pain, and sleep disorders [10,11]. the aetiology of sleep disorders in this patient group remains unresolved. there is no evidence that it is related to motor symptoms or that the relief from botulinum toxin treatment used for motor symptoms does not improve sleep disorders [12]. video-polysomnographic recordings in cd patients showed that the activity over cervical muscles disappeared during all sleep stages and thus could not influence sleep impairment [13]. one of the theories is that sleep disturbance is related to the dysfunction of some brain regions, such as the basal ganglia, with dopaminergic system disturbance. another theory is that it is related to depression [14]. parkinson's disease (pd) is a neurodegenerative disorder with alpha-synuclein inclusions as the main hallmark of disease pathology. during the disease course, α-synuclein pathology spreads from the brain stem to higher cortical regions, with consequential neuron degeneration. as a result of degeneration, there is a loss of many neurotransmitters in the brain, such as dopamine, serotonin, noradrenaline, and acetylcholine. this causes many motor and nonmotor symptoms, including sleep disorders, among many other non-motor symptoms described [15]. multiple factors could influence sleep disturbances, such as age-related seemedj 2020, vol 4, no. 2 sleep disorders in neurological diseases 37 southeastern european medical journal, 2020; 4(2) changes in sleep, nocturnal motor symptoms (rigidity, resting tremor, akinesia, tardive dyskinesia, and the ‘wearing-off’ phenomenon), non-motor symptoms (pain, hallucination, and psychosis), nocturia, and medication. besides that, as part of pd pathology, there are changes in the neurotransmitter systems (dopamine, norepinephrine, serotonin, and acetylcholine) responsible for regulating sleep structure and the sleep/wake cycle [16]. major depressive episodes are characterized by a period of depressed mood or anhedonia lasting for 2 or more weeks, with at least three additional signs, i.e. weight change or change in appetite, psychomotor agitation or retardation, feeling of worthlessness or guilt, diminished ability to concentrate, suicidal ideations or attempts, and insomnia or hypersomnia. changes in neurotransmitter levels, such as a decrease of serotonin, norepinephrine and dopamine, and hypercortisolaemia are possible aetiologic factors of depression. sleep disturbance is one of the most consistent symptoms. it can precede the symptoms of depression or persist after the disease remission and it is not related to depression itself. there are several theories on the aetiology of sleep disturbance in depression. one of them refers to monoaminergic level disruption, another one to glutamate level decrease, and one to hypercortisolaemia. however, none of them includes a clear conclusion regarding its aetiology [4]. the aim of the study was to assess the prevalence and characteristics of sleep disorders in patients with cervical dystonia, compared to healthy controls, patients with parkinson's disease, and patients with depression. subjects and methods in this cross-sectional study, we analysed 122 subjects, 30 of whom were healthy controls, 30 cd patients, 32 pd patients, and 30 were subjects diagnosed with depression. the study was conducted at the department of neurology and department of psychiatry, university hospital centre osijek, from february to may 2017. all the participants signed a written informed consent. the study protocol was reviewed and approved by the university hospital centre osijek ethics board and it was in accordance with the declaration of helsinki. cd patients were recruited from the botulinum toxin clinic and tested during their regular follow-up examinations (without relation to their botulinum injections schedule). the control group included sexand age-matched healthy relatives and friends of cd patients. pd patients were recruited from the movement disorders clinic. pd diagnosis was made according to the uk pd society brain bank (ukpdsbb) diagnostic criteria and both early and advanced pd patients were analysed [17]. depressed patients were recruited from the psychiatric ward where they were hospitalized due to depression problems. control, pd, and cd groups were tested for symptoms of depression and anxiety by using the beck depression inventory (bdi) and beck anxiety inventory (bai). severity of depression and anxiety were not evaluated in depressed patients. all patients were tested for sleep disturbances using the pittsburgh sleep quality index (psqi) for night-time disturbances and epworth sleepiness scale (ess) for daytime sleepiness problems. the psqi has been designed to assess sleep quality and disturbances over a 1-month time interval. it is a self-rated questionnaire that can be filled in 1015 minutes. there are 19 individual questions divided into 7 subscales assessing subjective sleep quality, sleep latency, sleep duration, habitual sleep efficiency, sleep disturbances, use of sleeping medication, and daytime dysfunction. the sum of all 7 subscales provides the global psqi score ranging from 0 to 21, where lower scores denote better sleep quality [18]. the ess is a simple self-administered questionnaire designed to measure daytime sleepiness. it consists of 8 questions about how likely would it be for the subject to doze off in 8 different situations (sitting and reading; watching tv; sitting, inactive, in a public place; as a passenger in a car for an hour without a break; lying down to rest in the afternoon; sitting and talking to someone; sitting quietly after lunch without alcohol; and, in a car, while stopped for seemedj 2020, vol 4, no. 2 sleep disorders in neurological diseases 38 southeastern european medical journal, 2020; 4(2) a few minutes in the traffic). after scoring every situation on a scale from 0 (would never doze off) to 3 (high chance to doze off), the total sum is calculated. the maximum score is 24. the score is higher in patients having more problems with daytime sleepiness [19]. a demographic questionnaire was designed for the study. all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards. study was approved in 2016 by local ethical committee of medical school in university of j. j. strossmayer in osijek. informed consent was obtained from all individual participants included in the study. statistical analysis categorical data were expressed as absolute frequencies and percentages, while the differences between the groups were tested by fisher’s exact test. numerical data were expressed as median and interquartile ranges or as mean and standard deviations, depending on whether the data indicated normal distribution, which was tested by the kolmogorov-smirnov test. correlation between variables that did not indicate normal distribution was tested with spearman's rank correlation coefficient. differences between the groups in which the data did not indicate normal distribution were tested by the kruskal-wallis test. statistical significance was defined as α=0.05. post hoc analysis of differences between the two groups was done with the mann-whitney u test and, after the bonferroni correction, statistical significance was defined as p<0.016. statistical analysis was conducted by using statistica 13 (statsoft inc., tulsa, oklahoma, usa). results there were differences among the patient groups in terms of age (pd patients were older than subjects from other groups), while the study groups were quite homogeneous in terms of sex (table 1). there was no difference in disease duration among the three groups of subjects (table 2). table 1 demographic data regarding sex and age control parkinson’s disease focal dystonia depression p n/% n/% n/% n/% sex  male  female 10 (33.3) 20 (66.7) 20 (62.5) 10 (37.5) 10 (33.3) 20 (66.7) 11 (36.7) 19 (63.3) 0.052* age  20-30  31-40  41-50  51-60  61-70  71-80 1 (3.3) 2 (6.7) 3 (10.0) 10 (33.3) 8 (26.7) 6 (20.0) 0 (0) 0 (0) 3 (9.4) 3 (9.4) 18 (56.3) 8 (25.0) 1 (3.3) 2 (6.7) 3 (10.0) 10 (33.3) 8 (26.7) 6 (20.0) 0 (0) 3 (10.0) 4 (13.3) 16 (53.3) 4 (13.3) 3 (10.0) 0.004* *fisher’s exact test seemedj 2020, vol 4, no. 2 sleep disorders in neurological diseases 39 southeastern european medical journal, 2020; 4(2) table 2 differences in disease duration, symptoms of depression and anxiety between groups control parkinson’s disease focal dystonia depression median (iqr) mean median (iqr) mean median (iqr) mean median (iqr) mean p disease duration 5.00 (2.00-9.75) 6.31 7.00 (2.25-12.25) 7.20 7.00 (2.75-15) 9.42 0.315† bdi 1.00 (1.00-1.25) 1.37 2.00 (1.00-3.00) 2.19 1.50 (1.00-2.25) 1.83 0.020† bai 1.00 (1.00-1.00) 1.13 1.00 (1.00-2.00) 1.56 1.00 (1.00-2.00) 1.37 0.021† bdi – beck depression inventory; bai – beck anxiety inventory; †kruskal-wallis test post hoc analysis performed between groups by applying the mann-whitney u test indicated to the following significant differences: bdi between control and pd group (p < 0.008); bai between control and pd group (p < 0.006). there were significant differences in bdi and bai scores between control, pd, and cd groups (bdi p<0.020 and bai p<0.021). the post hoc analysis conducted between the groups by using the mann-whitney u test and the bonferroni correction indicated a significant difference in bdi and bai only between the pd and the control group (table 2). a positive correlation between the bdi and psqi scores in both patient groups (cd group rs 0.409, p<0.025; pd group rs 0.668, p<0.001) was found, but not between bdi and ess (cd group rs 0.191, p<0.312; pd group rs 0.093, p<0.612). when correlating bai and psqi, positive correlations both for pd and cd groups (pd group rs 0.604, p<0.001; cd group rs 0.370, p<0.044) was found, whereas for bai and ess a positive correlation only for the cd group was found (cd group rs 0.393, p<0.032; pd group rs 0.271, p<0.133). the frequency of severity in the psqi scale, ess, and psqi subjective score are shown in table 3. table 3 frequency of severity of sleep disorders and subjective assessment between groups control parkinson’s disease focal dystonia depression n/% n/% n/% n/% p ess  lower normal ds  higher normal ds  mild excessive ds  moderate excessive ds  severe excessive ds 17 (56.7) 9 (30.0) 2 (6.7) 2 (6.7) 0 (0) 11 (34.4) 9 (28.1) 2 (6.3) 8 (25.0) 2 (6.3) 19 (63.3) 6 (20.0) 3 (10.0) 1 (3.3) 1 (3.3) 14 (46.7) 10 (33.3) 1 (3.3) 1 (3.3) 4 (13.3) 0.035* psqi  normal  poor sleep quality 23 (76.7) 7 (23.3) 7 (21.9) 25 (78.1) 10 (33.3) 20 (66.7) 0 (0) 30 (100) 0.001* psqi subjective  very good  fairly good  fairly bad  very bad 14 (46.7) 11 (36.7) 3 (10.0) 2 (6.7) 9 (28.1) 8 (25.0) 11 (34.4) 4 (12.5) 8 (26.7) 12 (40.0) 7 (23.3) 3 (10.0) 3 (10.0) 10 (33.3) 11 (36.7) 6 (20.0) 0.004* ds – daytime sleepiness; ess – epworth sleepiness scale; psqi – pittsburgh sleep quality index; * fisher’s exact test seemedj 2020, vol 4, no. 2 sleep disorders in neurological diseases 40 southeastern european medical journal, 2020; 4(2) post hoc analysis showed the following differences between the groups: control and cd (psqi score p < 0.001); pd and cd (ess score p < 0.017); pd and depression (psqi score p < 0.007); cd and depression (psqi score p < 0.001; subjective psqi p < 0.042) we found significant differences among the groups for both scales and subjective psqi scores. post hoc analysis showed differences between the two groups (table 3). table 4 shows psqi subscale scores, while figure 1 shows global psqi scores for the groups. table 4 differences in psqi subscale scores between healthy control, parkinson disease (pd), focal dystonia (fd) and depression groups control parkinson’s disease focal dystonia depression median (iqr) median (iqr) median (iqr) median (iqr) p latency 0.00 (0.00-0.00) 1.00 (0.00-1.00) 1.00 (0.00-1.25) 2.00 (1.00-3.00) 0.001† effectiveness 0.00 (0.00-0.25) 1.00 (0.00-2.75) 0.00 (0.00-2.00) 2.00 (0.00-3.00) 0.001† duration 1.00 (0.00-2.00) 1.50 (0.00-2.00) 0.00 (0.00-0.00) 2.00 (0.00-3.00) 0.001† disturbances 0.00 (0.00-1.25) 0.00 (0.00-1.75) 2.00 (0.00-3.00) 2.00 (2.00-3.00) 0.001† awakening 1.00 (0.00-2.00) 3.00 (1.00-3.00) 2.50 (1.00-3.00) 3.00 (2.00-3.00) 0.001† toilet 1.00 (0.00-2.00) 3.00 (1.25-3.00) 2.00 (0.00-3.00) 3.00 (1.00-3.00) 0.001† breathing 0.00 (0.00-0.00) 0.00 (0.00-1.00) 0.00 (0.00-0.25) 0.00 (0.00-3.00) 0.043† snoring 0.00 (0.00-1.00) 2.00 (0.00-3.00) 0.00 (0.00-3.00) 3.00 (0.00-3.00) 0.001† cold 0.00 (0.00-0.00) 0.00 (0.00-1.75) 0.00 (0.00-0.00) 0.00 (0.00-3.00) 0.029† hot 0.00 (0.00-0.00) 0.00 (0.00-1.75) 0.00 (0.00-2.25) 2.00 (0.00-3.00) 0.079† nightmares 0.00 (0.00-0.00) 1.00 (0.00-2.00) 0.00 (0.00-1.25) 2.00 (0.00-3.00) 0.001† pain 0.00 (0.00-0.00) 2.00 (0.00-3.00) 2.00 (0.00-3.00) 1.00 (0.00-3.00) 0.001† other 0.00 (0.00-0.00) 0.00 (0.00-0.00) 0.00 (0.00-0.00) 3.00 (0.00-3.00) 0.001† hypnotics usage 0.00 (0.00-0.00) 0.00 (0.00-2.00) 0.00 (0.00-2.25) 3.00 (3.00-3.00) 0.001† dysfunctionality 0.00 (0.00-0.00) 0.00 (0.00-1.75) 0.00 (0.00-1.00) 2.00 (0.00-2.00) 0.001† vigilance 0.00 (0.00-0.00) 0.00 (0.00-1.00) 0.00 (0.00-0.00) 0.00 (0.00-1.25) 0.009† enthusiasm 0.00 (0.00-0.00) 0.00 (0.00-1.00) 0.00 (0.00-1.00) 3.00 (0.00-3.00) 0.001† psqi – pittsburgh sleep quality index; †kruskal-wallis test seemedj 2020, vol 4, no. 2 sleep disorders in neurological diseases 41 southeastern european medical journal, 2020; 4(2) post hoc analysis of differences between two groups carried out by applying the mann-whitney u test yielded significant differences for this subscale, and they are as follows: control and cd (p<0.001); pd and depression (p<0.001); cd and depression (p<0.001); duration score between control and cd (p<0.001); pd and cd (p<0.001); cd and depression (p<0.001); disturbance score between pd and depression (p<0.001); snoring score between cd and depression (p<0.009); nightmare score between control and cd (p<0.004); cd and depression (p<0.003); pain score between control and cd (p<0.001); other score between pd and depression (p<0.001); cd and depression (p<0.001); hypnotics usage score between pd and depression (p<0.001); cd and depression (p<0.001); dysfunctionality score pd and depression (p<0.006); cd and depression (p<0.001); enthusiasm score between pd and depression (p<0.001); cd and depression (p<0.001). the median score did not differ between some groups, but the interquartile range indicated to differences among them. significant differences among the groups in terms of global scores were observed (figure 1), but also with regard to many subscales. post hoc analysis, following the application of the bonferroni correction, showed differences between the two groups (table 4). figure 1 global psqi score between groups psqi – pittsburgh sleep quality index. post hoc analysis consisting of the mann-whitney u test, caried out after bonferroni correction in order to determine differences between two groups: psqi score between control and cd groups (p < 0.004); pd and depression groups (p < 0.001); and cd and depression groups (p < 0.001). seemedj 2020, vol 4, no. 2 sleep disorders in neurological diseases 42 southeastern european medical journal, 2020; 4(2) table 5 shows results pertaining to ess subscale scores, while figure 2 shows total ess scores for all the groups. there were no significant differences in total ess scores between the cd and control group, or between patient groups (after bonferroni correction) (figure 2). only the subscale “sitting in a public place” presented lower results in the pd group when compared to cd and depression groups (table 5). table 5 differences in ess subscale scores in between healthy control, parkinson disease, focal dystonia, and depression groups. control parkinson’s disease focal dystonia depression median (iqr) median (iqr) median (iqr) median (iqr) p watching tv 1.50 (1.00-2.00) 1.50 (1.00-2.00) 1.00 (0.00-2.00) 1.00 (0.00-2.25) 0.208† sitting, inactive, in a public place 0.00 (0.00-1.00) 1.00 (0.00-1.00) 0.00 (0.00-1.00) 0.00 (0.00-1.25) 0.147† passenger in a car 0.00 (0.00-1.00) 0.00 (0.00-2.00) 0.00 (0.00-1.00) 0.00 (0.00-1.00) 0.458† lying down to rest in the afternoon 2.00 (1.00-3.00) 2.00 (1.00-3.00) 2.00 (1.00-2.00) 2.00 (0.75-2.25) 0.402† sitting and talking 0.00 (0.00-0.00) 0.00 (0.00-1.00) 0.00 (0.00-0.25) 0.00 (0.00-1.00) 0.129† sitting after lunch 0.00 (0.00-1.00) 1.00 (0.00-3.00) 0.00 (0.00-1.00) 1.00 (0.00-2.00) 0.011† sitting in a public place 0.00 (0.00-0.00) 0.00 (0.00-1.00) 0.00 (0.00-0.00) 0.00 (0.00-0.00) 0.005† in a car, while stopped for a few minutes 0.00 (0.00-1.00) 1.00 (0.00-2.00) 1.00 (0.00-1.00) 0.00 (0.00-2.00) 0.390† ess – epworth sleepiness scale; †kruskal-wallis test post hoc analysis of differences between two groups carried out by applying the mann-whitney u test, which after applying the bonferroni correction (p<0.016) yielded significant differences for this subscale, and they are as follows: sitting after the lunch for pd and cd (p < 0.042); sitting in public for pd and cd (p < 0.013); pd and depression (p < 0.005). seemedj 2020, vol 4, no. 2 sleep disorders in neurological diseases 43 southeastern european medical journal, 2020; 4(2) figure 2 total ess scores between groups ess – epworth sleepiness scale. post hoc analysis consisting of the mann-whitney u test that was performed to determine the differences between two patient groups showed the following differences: ess score for pd and cd groups (p < 0.031). discussion patients with cd were analysed for sleep disturbance relative to the control group, and all three patient groups were analysed for sleep disturbances relative to each other. when compared to the control group, cd patients displayed more frequent sleep problems that included higher psqi scores. however, there was no difference with regard to daytime sleepiness problems. they have reported longer latency to fall asleep, lower duration of sleep during the night, more frequent nightmares, and more pain that disturbed their night-time sleep. avanzino et al. reported impairment in sleep duration, latency, and efficiency of sleep in patients with blepharospasm, but not in the cd patient group. likewise, antelmi et al. also found decreased sleep efficiency and increased sleep latency in video-polysomnographic recordings of cd patients [13,20]. impairment in sleep latency has its pathophysiological basis in delayed endogenous melatonin secretion, probably due to lower noradrenaline and serotonin levels that are an important part of this process [1]. although all three patient groups suffered from sleep disturbances, we still found differences among them. sleep of pd patients during the night is of shorter duration when compared to the cd group. pd group was older than the cd group, so this difference could be related to age, seemedj 2020, vol 4, no. 2 sleep disorders in neurological diseases 44 southeastern european medical journal, 2020; 4(2) i.e. it could be a consequence of age-related diminution in the output of the circadian pacemaker [7]. the depression group differed from the cd group in terms of psqi scores and most subscales. they reported prolonged latency, shorter duration of sleep, and worse quality of sleep resulting from snoring and nightmares. they also use hypnotics more often and are more likely to suffer from dysfunctionality and loss of enthusiasm during the day arising from their sleep problems. the depression group reported more of such similar problems when compared to the pd group (latency, duration of sleep, disturbances, and other problems and disturbances). they also used hypnotics more often and reported dysfunctionality and loss of enthusiasm during the day arising from their sleep problems. so, of the three patient groups, sleep problems were most pronounced in the depression group, followed by pd, and finally the cd group. smit et al. published a paper about altered presynaptic serotonin transporter (sert) binding in cd patients. they found that sleep disturbances were strongly linked to sert binding in the raphe nuclei in cd patients [21]. depression and anxiety in cd patients are related to serotonergic system impairment. patients with cd accompanied by depression and anxiety present lower sert (serotonin transport) binding in the midbrain/diencephalon [22]. we found that both anxiety and depression symptoms correlate with sleep disturbances in the cd group. this indicates that neurotransmitter impairment leading to depression and anxiety could be the aetiological factor of sleep disturbances in the cd group. all three patient groups had problems with waking up and there was no significant difference among them. dopamine-containing neurons are involved in the regulation of the waking process [23]. there is evidence that the dopaminergic system is disturbed in cd patients with depression. zoons et al. found alterations of striatal dat (dopaminergic transport) and d2/3 receptor binding in cd patients with depression [24]. in addition to dopamine, noradrenaline and acetylcholine are also important in the waking process. noradrenaline levels are high during the waking state and low during sleep [25]. noradrenergic and cholinergic cells in the pons increase firing to activate the waking pattern [26,27]. there is a case report about pathologic findings from an autopsy of a patient with primary segmental dystonia (meige syndrome). the authors found moderate-to-severe neuronal loss in several brainstem nuclei, including the substantia nigra pars compacta, locus coeruleus, raphe nuclei, and pedunculopontine nucleus [28]. another paper with histopathologic findings in cd patients did not report a neuronal loss in the pedunculopontine region, but described a pedunculopontine nucleus choline acetyltransferase deficiency with a functional cholinergic deficit [29]. this could explain the lower noradrenaline and acetylcholine levels in cd patients, which are an important part of the wake-sleep cycle. there was no difference in ess scores between the cd and control group, but the pd group reported lower ess scores more frequently when compared to the cd patient group. in terms of subscales, and after the bonferroni correction, a significant difference was found only with regard to the risk of falling asleep while sitting in public, where pd patients reported higher chances of falling asleep when compared to the cd and depression group. although excessive daytime sleepiness has not been indicated in cd patients [10,20,30], trotti et al. reported opposite results for their cd patients when compared to the control group. they explained that this could be attributed to the use of anticholinergics that can affect sleepiness during the day in a certain percentile of patients [14,32]. there is no evidence that impairment of sleep quality has an impact on daytime sleepiness [19,32]. conclusion sleep disturbances are frequent non-motor symptoms in cd, pd, and depression patients. according to our data, all three groups of patients had sleep disorders, but they differed in the frequency and extent of those disorders, with less pronounced symptoms found in cd seemedj 2020, vol 4, no. 2 sleep disorders in neurological diseases 45 southeastern european medical journal, 2020; 4(2) and pd patients. symptoms of depression and anxiety correlate with sleep disturbances in pd and cd patient groups. cd patients do not experience daytime sleepiness problems. the aetiology of sleep disturbances in cd patients is probably related to monoamine neurotransmitter system impairments similar to those of pd patients, but to a lesser extent than in patients with depression. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. zisapel n circadian rhythm sleep disorders pathophysiology and potential approaches to management. cns drugs 2001; 15(4):311-28. 2. gallopin tfp, eggermann e, caull b, luppi ph, rossier j, audinat e, mühlethaler m, serafin m. identification of sleep promoting neurons in vitro. nature 2000; 404:992-5. 3. pace-schott ef, hobson ja. the neurobiology of sleep: genetics, cellular physiology and subcortical networks. nat rev neurosci 2002; 3(8):591-605. 4. murphy mj, peterson mj. sleep disturbances in depression. sleep med clin 2015; 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258(10):1835-40. 31. yang j, shao n, song w, wei q, ou r, wu y, shang h-f. nonmotor symptoms in primary adult-onset cervical dystonia and blepharospasm. brain behav 2016; 7(2):e00592. trotti lm, esper cd, feustel pj, bliwise dl, factor sa. excessive daytime sleepiness in cervical dystonia. parkinsonism relat disord 2009; 15(10):784-6. i seemedj 2020, vol 4, no. 2 sleep disorders in neurological diseases 47 southeastern european medical journal, 2020; 4(2) i author contribution: acquisition of data: tomic s, degmecic d, gjoni f, dumencic i, milanovic s, gilman kuric t, popovic z, mirosevic zubonja t administrative, technical or logistic support: tomic s, degmecic d, gjoni f, dumencic i, milanovic s, gilman kuric t, popovic z, mirosevic zubonja t analysis and interpretation of data: tomic s, degmecic d, gjoni f, dumencic i, gilman kuric t, popovic z, mirosevic zubonja t conception and design: tomic s, degmecic d, gjoni f, dumencic i, gilman kuric t, popovic z, mirosevic zubonja t critical revision of the article for important intellectual content: tomic s, degmecic d, gjoni f, gilman kuric t, popovic z, mirosevic zubonja t drafting of the article: tomic s, degmecic d, gjoni f, dumencic i, gilman kuric t, popovic z, mirosevic zubonja t final approval of the article: tomic s, degmecic d, gjoni f, dumencic i, popovic z, mirosevic zubonja t guarantor of the study: tomic s, degmecic d, gjoni f, dumencic i, popovic z, mirosevic zubonja t provision of study materials or patients: degmecic d, gjoni f, dumencic i, gilman kuric t, popovic z, mirosevic zubonja t statistical expertise: tomic s, degmecic d, gjoni f, dumencic i, popovic z, mirosevic zubonja t seemedj 2021, vol 5, no. 2 characteristics and outcomes of patients with deep vein thrombosis 18 southeastern european medical journal, 2021; 5(2) original article characteristics and outcomes of patients with deep vein thrombosis diagnosed in emergency department of clinical hospital dubrava during 20191 ivan radilj 1, vlatko grabovac 1,2, zdravko mitrović *1,3 1 school of medicine, university of zagreb, zagreb, croatia 2 department of emergency medicine, clinical hospital dubrava, zagreb, croatia 3 division of hematology, department of medicine, clinical hospital dubrava, zagreb, croatia *corresponding author: zdravko mitrović, zdravko.mitrovic@kbd.hr received: aug 31, 2021; revised version accepted: nov 8, 2021; published: nov 26, 2021 keywords: deep vein thrombosis, emergency department, novel oral anticoagulants, rivaroxaban, warfarin, bleeding abstract aim: deep vein thrombosis (dvt) is a common clinical condition encountered in the emergency department (ed). the aim of this study was to compare the characteristics and outcomes of patients with respect to treatment using novel oral anticoagulants (noac). materials and methods: in this retrospective observational study, we analyzed medical records of patients diagnosed with dvt during 2019 in the ed of the clinical hospital dubrava. we identified 295 patients, who comprised 1.2% of all patients examined in the ed. results: women were more frequently diagnosed with dvt (59%) and they were older than the men (median age 69 vs. 62 years, respectively). patients with proximal deep vein thrombosis (71%) were admitted to the hospital. two thirds of all patients were treated with noac. rivaroxaban was the most commonly prescribed drug (52% of patients). control doppler ultrasound was performed in 58% of the patients, and complete resolution was observed in 63% of the cases. noacs caused significantly fewer bleeding events than warfarin (3.2% vs. 13.6%, p < 0.05). conclusion: our results demonstrate that patients with dvt can be safely treated with noacs in an outpatient setting. (radilj i, grabovac v, mitrović z. characteristics and outcomes of patients with deep vein thrombosis diagnosed in emergency department of clinical hospital dubrava during 2019. seemedj 2021; 5(2); 18-26) seemedj 2021, vol 5, no. 2 characteristics and outcomes of patients with deep vein thrombosis 19 southeastern european medical journal, 2021; 5(2) introduction deep vein thrombosis (dvt) is a clinical condition diagnosed in the emergency department (ed) on a daily basis. the average annual incidence rate of venous thromboembolism (vte) in europeans ranges from 1.04 to 1.83 per 1,000 person-years (1). separately, pulmonary embolism (pe) with or without dvt varies from 0.29 to 0.78 per 1,000 person-years. dvt incidence rate ranges from 0.45 to 1.17 per 1,000 person-years. the total age-adjusted incidence rate is higher for men (1.3 per 1,000 person-years) than women (1.1 per 1,000 person-years) (1). the incidence rate in the population younger than 45 is 0.12 per 1,000 person-years, and 2.62 per 1,000 person-years in the population older than 65(2). in fact, dvt primarily affects older people. dvt occurs as a result of three overlapping mechanisms, known as virchow’s triad: venous stasis, endothelial injury and hypercoagulability. the most important one is venous stasis, but it is not sufficient for thrombus formation in and of itself. venous valve pockets are places of reduced blood flow in which thrombi develop. as a result of the blood flow slowing down, partial pressure of oxygen declines and consequently leads to local hematocrit increase. higher expression of prothrombotic and lower expression of antithrombotic proteins further enhance the hypercoagulable microenvironment. intact endothelial surface has antithrombotic and anticoagulant characteristics. endothelial dysfunction or injury promotes contact of venous blood with tissue factor and thrombin, which activates the coagulation cascade (3, 4). in croatia, according to the study conducted by the croatian cooperative group for hematologic diseases (krohem) in 2011, the estimated annual incidence of vte was 1.185 per 1,000 people (5). the incidence of dvt was 0.79 per 1,000 people, with female predominance (56.3%). there were more patients with secondary vte (57.3%), and malignant disease was the most frequent cause. since 2011, novel oral anticoagulants (noac) have emerged as a novel treatment option, in addition to low-molecular-weight heparin (lmwh) and vitamin k antagonists (vkas). three noacs were available in croatia in 2019, however, only with partial reimbursement by national insurance: dabigatran (prothrombin inhibitor), rivaroxaban and apixaban (factor xa inhibitors). other than venous thromboembolism, indications for noacs are atrial fibrillation and prevention of thromboembolic complications after a stroke or myocardial infarction. their use is simpler, with fixed oral dosing, there is no need for laboratory monitoring and they do not depend on the diet. patients can be treated with rivaroxaban and apixaban without lmwh as the standard initial therapy for dvt. apixaban and dabigatran require dosing twice per day, as opposed to rivaroxaban, which requires use once per day (6). the aim of this study is to determine the clinical characteristics, treatments and outcomes of patients who were diagnosed in the ed of the clinical hospital during 2019. patients and methods study design this study was designed as a retrospective analysis of electronic hospital charts of patients diagnosed with dvt in the emergency department of the clinical hospital dubrava, zagreb, croatia, during 2019. patients and definitions a total of 399 patients with vte were diagnosed from 1 january to 31 december 2019. patients that presented with pe without proven dvt were not included. medical records of the remaining 295 patients with dvt were analyzed. diagnosis was established based on clinical examination, d-dimer test and doppler ultrasound. the following data were collected: age, sex, date of diagnosis, thrombosis type (proximal or isolated distal deep vein thrombosis; or other thrombosis), admission to hospital, treatment, idiopathic or secondary dvt. dvt was seemedj 2021, vol 5, no. 2 characteristics and outcomes of patients with deep vein thrombosis 20 southeastern european medical journal, 2021; 5(2) considered secondary if some of the following risk factors were identified: malignant disease within 6 months of the event; recent trauma, surgery, immobilization/immobility; use of oral contraceptives; pregnancy; inflammatory bowel disease and thrombophilia. dvt was considered idiopathic if none of above factors were present. in our study, bleeding was considered significant if a patient required examination in the emergency department. statistical analysis categorical data are presented with absolute (n) and relative frequencies and compared using the χ2 test or fisher’s exact test, where applicable. numerical data are presented with median and range values and compared using the mann-whitney u test. the probability value of < 0.05 was considered statistically significant. estimated annual incidence per 1,000 inhabitants was calculated using the number of recorded new dvt cases divided by the population which gravitates to this hospital. microsoft excel was used for statistical analysis. ethics our research was conducted in full compliance with the declaration of helsinki and it was approved by the ethics committee of the clinical hospital dubrava. results a total of 295 patients were diagnosed with dvt, and 104 were diagnosed with pe (without concomitant dvt) out of a total of 23,899 patients examined in the ed in 2019 (figure 1). figure 1. patient flowchart (total n = 23,899) ed = emergency department, vte = venous thromboembolism, pe = pulmonary embolism, dvt = deep vein thrombosis it should be noted that 55 dvt patients (13.8% of the total number) were diagnosed with concomitant pe. thus, dvt frequency in our ed was 1.23% and pe frequency was 0.67%. total vte frequency was 1.67%. considering the number of people gravitating to the clinical hospital dubrava (which is about 330,000), the estimated annual dvt incidence was 0.894 per 1,000 people. median age was 65 years, ranging from 20 to 94 years. women comprised 59% of patients (174 out of 295). women were older (median 69 years; range 22 to 90) than men (median 62 years; range 20 to 94) (p = 0.005). however, in the younger age groups, the incidence of thrombosis was higher in men (figure 2).. seemedj 2021, vol 5, no. 2 characteristics and outcomes of patients with deep vein thrombosis 21 southeastern european medical journal, 2021; 5(2) figure 2. proportion of men and women with deep vein thrombosis according to age groups (total n = 295) admission to the hospital was required for 206 patients (69.8%), and 73 patients (24.8%) were treated in an outpatient setting, while 16 (5.4%) refused hospitalization or were sent to another hospital. the patients who were admitted to the hospital were older compared to those who were discharged from the ed (median age was 69 years (range 20 to 94) vs. 60 years (range 25 to 88), respectively) (p = 0.0002). proximal dvt (thrombosis of the popliteal vein and of the femoral veins) was diagnosed in 210 patients (71.2%), while isolated distal dvt was diagnosed in 72 patients (24.4%) (figure 1). of the 13 patients with other thrombosis, ivc thrombosis was diagnosed in four patients (1.4%), internal jugular vein thrombosis in two patients (0.6%) and thrombosis of arm veins in seven patients (2.4%). of the patients with leg thrombosis, left leg thrombosis occurred in 51.8% of patients compared to 46.5% of patients with right leg thrombosis (1.7% had thrombosis in both legs). a total of 67 patients (22.7%) had recurrent dvt. divided by gender, there were 37 women (21.3%) and 30 men (24.8%) with recurrent dvt (p = 0.38). table 1. secondary dvt* causes (total n = 170) dvt causes n (%) malignant disease 69 (40.6%) trauma, operation, immobilization 56 (32.9%) immobility 19 (11.2%) hormonal contraception 4 (2.4%) other 22 (12.9%) *dvt = deep vein thrombosis a provoking factor was identified in 170 patients (57.6%), while 125 patients (42.4%) did not have any factors in their medical documentation. patients with idiopathic dvt appear to be older (median age 67 years) than those with secondary thrombosis (median age 64 years) (p 20-29 30-39 40-49 50-59 60-69 70-79 80+ women 2 9 23 14 42 54 30 men 8 9 14 21 29 24 16 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% men women seemedj 2021, vol 5, no. 2 characteristics and outcomes of patients with deep vein thrombosis 22 southeastern european medical journal, 2021; 5(2) = 0.435). the most common provoking factor in the group of secondary dvt patients was active malignant disease in 69 patients (40.6%) (table 1). based on gender, there were 38 women (37.6%) and 31 men (44.9%) with malignant disease as a provoking factor (p = 0.34). recent trauma, surgical procedure or limb immobilization was present in 56 patients (32.9%). there were 19 immobile patients (11.2%), while four patients (2.4%) were using oral contraceptives. the remaining 22 patients (12.9%) with secondary dvt had inflammatory bowel disease, thrombophilia, were pregnant, or failed to adhere to anticoagulant treatment after previous vte. initial treatment with lmwh was started in 264 patients (90.7%). the most frequent treatment after initial lmwh was rivaroxaban in 130 patients (44.1%) (table 2). rivaroxaban without previous lmwh therapy was used in 24 patients (8.1%). the second most frequent therapeutic option was warfarin after lmwh, in 59 patients (20%). long-term lmwh therapy was the option of choice for 46 patients (15.6%), mainly for patients with active malignant disease. dabigatran or apixaban after initial lmwh treatment was used in 31 patients (10.5%). acenocoumarol was the treatment option for one patient (0.3%). therapy for four patients (1.4%) is unknown due to their transfer to another hospital. table 2. treatment modalities (total n = 295) therapy n (%) rivaroxaban after starting lmwh* 130 (44.1%) rivaroxaban without previous lmwh 24 (8.1%) warfarin 59 (20%) long-term lmwh 46 (15.6%) dabigatran/apixaban 31 (10.5%) acenocoumarol 1 (0.3%) unknown 4 (1.4%) *lmwh = low-molecular-weight heparin we also compared the bleeding in patients on noac and warfarin. of the 185 patients treated with noac, there were only six significant bleeding events (3.2%) that required medical attention in the ed. in comparison, of the 59 patients treated with warfarin, eight bleeding events were documented (13.6%). this is a statistically significant difference; p = 0.0067. the median follow-up period for all patients was 260 days (range 3 to 648 days), with 45 (15.3%) patients lost to follow-up. patients treated with warfarin had a similar median follow-up period compared to those treated with noac, 237 days (range 6 to 613 days) and 280 days (range 3 to 648 days), respectively. a follow-up doppler ultrasound was performed in 170 patients (57.6%); 116 patients (39.3%) did not come for a follow-up examination in our institution, 6 (2.1%) were treated in another hospital and 3 patients (1%) died during or soon after hospitalization. complete resolution was confirmed in 107 patients (62.9%), partial resolution was found in 60 patients (35.3%), and 3 patients (1.8%) did not show any improvement. of the 129 patients treated with noac with available follow-up, 80 patients showed complete resolution (62%), whereas of the 31 patients treated with warfarin, 17 patients showed complete resolution (55%); p = 0.10. discussion this study provided a valuable insight into the clinical characteristics and treatment of patients with dvt in croatia in the era of noacs. estimated annual incidence of vte from this research is consistent with other published studies (1, 2, 5). vte frequency in the ed of the clinical hospital dubrava is comparable to the seemedj 2021, vol 5, no. 2 characteristics and outcomes of patients with deep vein thrombosis 23 southeastern european medical journal, 2021; 5(2) study conducted by krohem, and is almost identical to the spanish study (1.67% vs. 1.65%) (7). an interesting finding of this study is that dvt was diagnosed more frequently in women. in this study, we had 59% of women with dvt, which is comparable to other studies (java, krohem) (5, 8). according to the estimation by the croatian bureau of statistics, the proportion of women in the general population in 2018 was 51.7%, with increasing numbers in older age groups, and the average life expectancy of women in croatia is 7 years longer than that of men (10). however, in patients younger than 65, there were only 48.6% of women (figure 2). overall, it seems that the female sex is not an independent risk factor for dvt. interestingly, there was a higher rate of simultaneous dvt and pe (13.8%) than in the study conducted by krohem (5.4%), while the japan vte treatment registry (java) reported a rate of 14.4% (8). a reason for such difference could be the more frequent use of pulmonary angiography compared to the general hospitals included in the krohem study eight years prior to this study. it is known that with proximal dvt, asymptomatic pulmonary embolism can occur in 35% of the cases (9). conversely, in patients with pe, deep vein thrombosis can be diagnosed in up to 71% of all patients (10). it is important to note that the ratio between proximal and distal dvt in our study is comparable to other studies, such as the norwegian study, which reported 69.6% of patients with proximal dvt and 27.2% of patients with distal dvt (2). almost two thirds of the patients in 2019 were treated with noacs, and one quarter of the patients were treated in an outpatient setting. also, a substantial finding of this study is that noacs cause fewer bleeding events compared to warfarin. introducing noacs into everyday clinical practice enabled a higher rate of outpatient management. for instance, in a spanish study conducted in 2002, 99% of patients with pe and 85% of patients with dvt were admitted to the hospital (12). a more recent study from spain, conducted in 2014 in 53 eds across spain, reported that 98.7% of patients with pe and 50.2% of patients with dvt were hospitalized (7). in our study, 71% of the patients were admitted to the hospital. this fully corresponds to the proportion of patients with proximal dvt. in other words, patients with distal dvt were treated in an outpatient setting using noacs. the prevalence of dvt on the left side (52.7% vs. 47.3%) found in our study was also observed in other studies (13, 14). the only valid explanation for that difference is compression of the left common iliac vein by the right common iliac artery (may-thurner syndrome) (13). the proportion of idiopathic venous thrombosis in our cohort (42.4%) is consistent with other studies. idiopathic thrombosis was diagnosed in 43.2% and 42.7% of the patients in the java and the krohem studies, respectively (5, 8). both the krohem study and this study report on the greater age of patients with idiopathic thrombosis compared to patients with secondary thrombosis. venous thromboembolism is a well-known complication of malignant disease. in a recent study conducted on 1,041 patients with solid tumors, 7.8% of patients had a thromboembolic event (15). both the krohem study and this study recognized malignant disease as the leading cause of secondary thrombosis. the second most common cause (trauma, surgical procedure and immobilization) was less frequent in this study (32.9%) compared to the krohem study (38.2%). inability to move as a result of paralysis or frailty is considered to be the third most common risk factor. oral hormonal contraception (ohc) is an important risk factor in the population of fertile women (15– 45 years). of the 16 fertile women with secondary thrombosis, 4 had ohc in their medical history (25%). the riete study reported that 36% of women with vte younger than 50 years were using ohc (16). other risk factors were less frequent (table 1). initial treatment with lmwh was used in 94% of patients included in the spanish study (7). that percentage is comparable to this study (90.7%). there were 12,585 patients with the first vte episode included in a large norwegian study, which excluded patients with malignant disease (17). rivaroxaban was a treatment option in 46.3% seemedj 2021, vol 5, no. 2 characteristics and outcomes of patients with deep vein thrombosis 24 southeastern european medical journal, 2021; 5(2) of patients, warfarin in 28.3%, apixaban in 24.5% and dabigatran in 0.7%. this study included patients with malignant disease, of whom two thirds were treated using prolonged lmwh therapy. considering the entire dvt population, the most commonly used therapeutical option was rivaroxaban (52.2%) (table 2). it is known that outpatient management using rivaroxaban can be efficiently performed in low-risk patients, with a low rate of bleeding and recurrent thrombotic events (18). other noacs were less commonly prescribed (10.5%). this may be explained by the fact that dabigatran requires five days of prior lmwh treatment, which is complicated in an outpatient setting, as well as by the fact that apixaban was introduced in croatia several years after rivaroxaban. on the other hand, warfarin was prescribed to 1 in 5 patients (table 2). it is obvious that noacs are the preferred choice for an increasing number of patients. in addition, patients report greater satisfaction with noacs compared to warfarin (19). one of the reasons for that is the lower incidence of bleeding when using noacs (20), which was confirmed in this study. we observed statistically fewer serious bleeding events in patients treated with noacs compared to those treated with warfarin (table 3). table 3. bleeding during treatment with noac* and warfarin (total n = 244) n bleeding incidence warfarin 59 8 13.6% noac 185 6 3.2% *noac = novel oral anticoagulants deep vein thrombosis is a disease which requires regular check-ups because of the high incidence of recurrent thrombosis. the five-year cumulative incidence of recurrent vte events is 21.5% after the first dvt episode and 27.9% after the second (21). we had 22.7% patients with recurrent dvt after previous vte. as a comparison, in the study conducted by krohem, recurrent vte was diagnosed in only 11.9% of patients (5). however, as many as 39% of the patients diagnosed with dvt in our cohort in 2019 have had no further ultrasonographic check-ups in our institution. this could be explained by the fact that clinical hospital dubrava was the national covid-19 hospital during a few months in 2020 and 2021 and the patients were probably followed-up on in other institutions. data about check-ups (and complications) observed in other institutions are not available. that is the main weakness of our study. nevertheless, complete dvt resolution was documented in almost two thirds of our patients. there was a trend toward better efficacy of noac compared to warfarin; 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13:1363–73. doi: 10.2147/ppa.s204246. 20. wadhera rk, russell ce, piazza g. warfarin versus novel oral anticoagulants. circulation. 2014; 130(22): e191–3. doi: 10.1161/circulationaha.114.010426 21. hansson po, sörbo j, and eriksson h. recurrent venous thromboembolism after deep vein thrombosis: incidence and risk factors. arch internal med. 2000; 160(6): 769–74. https://doi.org/10.1001/archinte.160.6.769.. 1 author contribution. acquisition of data: radilj i, grabovac v, mitrović z administrative, technical or logistic support: radilj i, grabovac v, mitrović z analysis and interpretation of data: radilj i, grabovac v, mitrović z conception and design: radilj i, grabovac v, mitrović z critical revision of the article for important intellectual content: radilj i, grabovac v, mitrović z drafting of the article: radilj i, grabovac v, mitrović z final approval of the article: radilj i, grabovac v, mitrović z provision of study materials or patients: radilj i, grabovac v, mitrović z statistical expertise (statistical analysis of data): radilj i, grabovac v, mitrović z seemedj 2021, vol 5, no. 2 blood usage and wastage at a public hospital setting 68 southeastern european medical journal, 2021; 5(2) original article effectiveness of initiatives to minimize blood usage and wastage at a public hospital setting in guyana 1 rajini kurup *1, audrey anderson 1, ramona bisnauth 1, shondel pompey-atkins 1, cecil boston 1, pheona mohamed-rambarran 2 1 college of medical sciences, university of guyana, georgetown, south america 2 georgetown public hospital cooperation, georgetown, guyana, south america *corresponding author: rajini kurup, rajini.kurup@uog.edu.gy received: oct 16, 2021; revised version accepted: nov 11, 2021; published: nov 26, 2021 keywords: blood wastage, donors, effectiveness, guyana abstract objectives: this is a cross-sectional descriptive follow-up study which analysed the pattern of blood usage and wastage after initiatives were taken following the initial study done in guyana from 2012 to 2014. this study also assessed the healthcare personnel’s knowledge regarding blood transfusion. methods: a study was conducted concerning blood product usage and wastage using data from the laboratory blood bank information system in 2016–2018 in the public hospital. information on knowledge, attitude, practices and administrative guidance of healthcare personnel was assessed using a self-administered questionnaire on different areas of transfusion medicine. usage of blood products was calculated as a percentage, and wastage of blood products was calculated as the number of units wasted due to each reason divided by the total number of units wasted. the data were entered and analysed in spss 21.0. results: a total of 29,577 units of blood were issued by the national blood transfusion service. each year, a blood unit collection of 9,745 (32.9%), 9,765 (33.0%), 10,067 (34.0%) units, respectively, was recorded. data indicated that 3,851 units (13.0%) of blood were wasted at the georgetown public hospital cooperation due to various reasons. packed red blood cells were the most commonly used blood product that was issued (52.5%) and platelets (47.8%) were the most commonly wasted product. in comparison to the previous study, blood wastage decreased from 25.4% to 13.0% after implementing simple interventions. results of examination of knowledge, attitude, practices and administrative guidelines of health personnel were not satisfactory. conclusion: simple and relatively cheap interventions introduced following the previous study had a dramatic impact on reducing blood wastage in the public hospital in guyana. (kurup r, anderson a, bisnauth r, pompey-atkins s, bostona c mohamed-rambarranb p. effectiveness of initiatives to minimize blood usage and wastage at a public hospital setting in guyana. seemedj 2021; 5(2); 68-76) seemedj 2021, vol 5, no. 2 blood usage and wastage at a public hospital setting 69 southeastern european medical journal, 2021; 5(2) introduction ensuring and practising safe and rational blood transfusion is important for high-quality and effective patient care in hospitals. the world health organization (who) defines blood management as “a patient-focused, evidencebased and systematic approach to optimize the management of patient and transfusion of blood products for quality and effective patient care. it is designed to improve patient outcomes through the safe and rational use of blood and blood products and by minimizing unnecessary exposure to blood products” (1). however, providing a safe and adequate supply of blood and blood products for transfusion is always expensive. appropriate clinical use of blood is an important aspect of blood safety and it reduces unnecessary exposure of patients to allogeneic blood and its associated risks (2). despite extensive use in emergency settings, blood transfusion decisions are always made without reasonable training and with limited knowledge of the patients’ situation. overuse or inappropriate use of blood products leads to inadequacy of blood components and a substantial increase in the cost of care. although blood components are a precious resource, their wastage is a critical problem in hospitals, especially in developing countries (3). blood and blood components are extremely valuable and require careful allocation to maximize clinical benefits. who reports that “[t]here are currently no global standards for the estimation of national requirements for blood and blood products. the need for blood and blood products is dynamic and is dependent on many factors related to health service coverage, the level of development and sophistication of the health care system and hospital blood usage” (2). there are multiple causes of blood wastage in hospitals, but the two major ones are: 1) delay in returning blood products after ordering and 2) exceeding shelf life (expiry) (3). as per the us food and drug administration (fda) and american association of blood banks (aabb) guidelines, the criterion for accepting issued blood products is that the product was kept within a temperature range of 1–6 °c (4, 5). in most countries, including guyana, blood banks follow a 30-minute rule for returning red blood cell (rbc) units. if the ordered rbc units are returned to the blood bank after 30 minutes without a controlled temperature, they must be discarded (6-8). therefore, wastage occurs if the product has not been transfused or returned within 30 minutes of its issue. in a previous study done in guyana on blood wastage and usage, it was found that 25% of blood was wasted due to various reasons (9). this study identified a large percentage of blood wastage in the only tertiary hospital in the country. in an ideal setting, expiry and wastage of blood products should not occur, although a very low level of expiry of blood products could be expected due to blood product stocks and unpredictable demands on inventory (10). moral responsibilities of healthcare providers, along with strict interventions and awareness, could reduce wastage of blood products (11). blood product wastage could be reduced through simple and cheap interventions (12). a study done on blood wastage by heitmiller et al. indicated that rbc wastage could be decreased by 61% over 4 years, saving more than $800,000 (13). simple interventions like strict management of blood stocks, tracking of released blood products and awareness of blood usage among clinical staff were introduced after the first similar study in the same setting. transfusion guidelines among clinicians could also increase awareness of blood transfusion. this study aimed to evaluate the efficiency of guidelines/interventions implemented after the previous study on blood usage and wastage. the interventions implemented after the first study helped in creation of awareness among hospital staff, creation of a proper electronic inventory and maintaining of proper temperature for blood and blood products. in order for the health professionals to make appropriate decisions in regard to proper management of blood products, it is very seemedj 2021, vol 5, no. 2 blood usage and wastage at a public hospital setting 70 southeastern european medical journal, 2021; 5(2) important to educate them regarding the risks and benefits of transfusion. this study also aimed to investigate the adequacy in terms of knowledge, attitude and practices among doctors, nurses and laboratory technologists in the public hospital. materials and methods this was a cross-sectional, descriptive study conducted between 2016 and 2018 in the only referral hospital in guyana. it was a follow-up study aimed at analysing the effectiveness of initiatives for minimizing blood usage and wastage in a public hospital setting in guyana. georgetown public hospital cooperation (gphc) is faced with high demand for blood for transfusion. national blood transfusion service (nbts), guyana, is responsible for ensuring and providing an adequate supply of blood components. blood collection, processing and screening of blood from donors is performed at the blood bank of the nbts. it is a centralized blood centre which collects blood directly or through blood drives from voluntary blood donors. nbts also collects blood through its blood facilities at the new amsterdam, west demerara, suddie and linden hospitals, along with mobile units for blood camps. this study was conducted in three phases to meet the objective of the study: a) to assess the usage and wastage of blood and blood products; b) to investigate the knowledge, attitude and practices (kap) of the health personnel; and c) to compare the usage and wastage of blood products with the data obtained in previous study. usage and wastage of blood and blood products wastage as a percentage of units issued (wapi) was calculated for red blood cells (rbc), platelets (plt), fresh frozen plasma (ffp), prethawed ffp (ptffp), pediatric packed cell (pedpc), thawed fresh frozen plasma (tffp) and packed red blood cell (prbc ): wapi = sum of wasted units for each component  100 sum of units issued for each component to the hospital percentage of blood and blood product wastage was calculated using the following formula: percentage = number of units wasted  100 total number of units wasted kap of health personnel a carefully formulated multiple choice questionnaire, identifying areas of knowledge, attitude and practices (kap) about blood usage and wastage, was prepared. the questions were adapted and modified from the aabb technical manual (17th edition), aabb standards for blood banks and transfusion services (27th edition) and other literature to focus on local interests. the questionnaire was divided into four sections to meet the objectives. the first part was on knowledge assessment, the second on attitude assessment, the third on practice assessment and the last on assessing administrative guidelines. the questionnaire took less than 15 minutes to complete. only doctors and nurses answered questions related to administrative guidelines. a score was given for each correct answer and converted into a percentage. mean scores were used to place each participant in a group: good or poor. participants who scored above the mean were considered to have good kap and those with a score below the mean were considered to have poor kap. the same applied to the independent components of kap. an overall average was calculated to compare the groups. a t-test and anova were used to compare a statistically significant difference in mean kap among the clinical staff involved. to validate and improve the questionnaire, a pilot study was conducted among a small group of clinicians and was not included in the actual study. data were first entered in ms excel and later analysed using spss 21.0. ethical considerations ethical approval for the study was obtained from the institution review board, ministry of public health, guyana and from the director of gphc before proceeding with the research. a signed seemedj 2021, vol 5, no. 2 blood usage and wastage at a public hospital setting 71 southeastern european medical journal, 2021; 5(2) consent form was obtained from all participants prior to their participation in the study. statistical analysis the data related to usage and wastage of blood products were presented as number, percentage, and standard deviation. the hi-2 test was used for statistical analysis and a pvalue less than 0.05 was considered statistically significant. results status of blood usage a total of 29,577 units of blood were collected in the referral hospital from the blood bank during the period from 2016 to 2018. for each year, a blood unit collection of 9,745 (32.9%; 95% ci 32.4– 33.5), 9,765 (33.0%; 95% ci 32.5–33.6), 10,067 (34.0%; 95% ci 33.5¬–34.6) units, respectively, was recorded. mean (± se) blood products collected annually were 331 ± 3.91 (95% ci 323.4– 338.7), 335 ± 4.43 (95% ci 326.3–343.7), 359 ± 4.33 (95% ci 350.5–367.5) for 2016, 2017 and 2018, respectively. compared to 2016, there was an increase in collection (by the referral hospital) of prbc, ffp, platelets and pedpc in 2018 by 4.7%, 6.1%, 16.6%, and 16.1%, respectively (figure 1). figure 1: distribution of blood products during the study period (2016–2018) of the blood products collected collectively and annually, the most frequent blood type was o+ with 50.6% (95% ci 50.0–51.2), followed by b+ (20.2%, 95% ci 19.7–20.6) and a+ (18.9%, 95% ci 18.4–19.3) (figure 2). usage of blood components during each quarter of the study period is shown in figure 3.. 0.0 10.0 20.0 30.0 40.0 50.0 60.0 prbc ffp platelets pedpc cryo ptffp tffp 51.7 18.6 13.1 5.2 3.6 3.9 3.8 58.4 18.7 12.2 6.2 2.6 3.4 1.8 52.5 19.2 15.2 6.0 2.7 1.9 2.4 2016 2017 2018 seemedj 2021, vol 5, no. 2 blood usage and wastage at a public hospital setting 72 southeastern european medical journal, 2021; 5(2) figure 2: distribution of blood groups during the study period (2016–2018) figure 3: blood components and their usage every quarter (2016–2018) status of blood wastage the wapi rates of cryo, ffp, ptffp, platelets, pedpc, tffp, and prbc were 17.2% (95% ci 14.7– 19.9), 15.9% (95% ci 14.9–16.9), 42.9 (95% ci 39.6– 46.2), 46.6 (95% ci 45.0–48.2), 5.0 (95% ci 4.0–6.1), 23.3 (95% ci 20.4–26.4) and 2.1 (95% ci 1.9–2.3), respectively. the highest wastage was observed with platelets, followed by ptffp and tffp. table 1 shows the various causes of blood wastage during the study period. the major reasons for significant blood wastage during the study period were expired blood unit (91.4%; 95% ci 90.4–92.2), broken bag (3.8%; 95% ci 3.2–4.4), unit returned after 30 minutes (1.0%; 95% ci 0.7– 1.4). 0.0 10.0 20.0 30.0 40.0 50.0 60.0 a+ ab+ bo+ oab+ ab18.6 1.2 17.7 1.1 53.3 2.9 4.9 0.3 19.0 1.2 20.9 1.3 50.4 3.0 3.9 0.2 18.9 1.2 21.8 1.0 48.2 3.4 5.0 0.4 2016 2017 2018 20.2 25.4 21.3 26.3 19.3 34.3 22.9 34.7 28.8 27.2 30.3 21.7 30.0 26.9 18.2 19.5 38.4 30.4 31.0 21.4 26.2 17.7 23.8 28.6 22.4 27.3 21.2 26.4 13.2 26.2 19.1 18.4 27.2 15.3 25.3 29.2 26.5 24.7 26.8 17.6 42.0 26.5 32.5 23.1 35.7 21.3 28.9 23.6 25.9 26.0 24.4 32.2 22.2 28.4 21.2 22.3 31.8 27.1 11.6 25.9 25.7 24.3 24.2 29.6 27.7 14.4 30.0 24.6 20.9 24.8 26.0 20.6 21.6 29.0 22.6 33.5 25.5 20.8 26.9 25.3 16.9 25.7 12.3 23.2 ffp plt tffp ffp plt tffp ffp plt tffp 2016 2017 2018 jan-mar apr-jun jul-sep oct-dec seemedj 2021, vol 5, no. 2 blood usage and wastage at a public hospital setting 73 southeastern european medical journal, 2021; 5(2) table 1: reasons for blood component wastage during the study period (2016–2018) reason for blood wastage 2016 2017 2018 total difference (%) pvalue expired unit 1301 (90.5) 1190 (91.6) 1028 (92.2) 3519 (91.4) 1.7 0.00 broken bag 72 (5.0) 34 (2.6) 40 (3.6) 146 (3.8) -1.4 0.0002 broken cold chain 21 (1.5) 28 (2.2) 19 (1.7) 68 (1.8) 0.2 0.4 return after 30 minutes 21 (1.5) 16 (1.2) 3 (0.3) 40 (1.0) -1.2 0.0 clotted blood 10 (0.7) 9 (0.7) 5 (0.4) 24 (0.6) -0.3 0.4 broken seal 8 (0.6) 9 (0.7) 2 (0.2) 19 (0.5) -0.4 0.1 component with rbc 3 (0.2) 4 (0.3) 0 7 (0.2) -0.2 0.2 expired transfusion unit 1 (0.1) 4 (0.3) 13 (1.2) 18 (0.5) 1.1 0.002 transfusion reaction 0 5 (0.4) 5 (0.4) 10 (0.3) 0.4 0.1 comparison of blood wastage with previous study figure 4 compares the blood wastage percentage recorded in the current three-year study and in the previous three-year study. each year showed a gradual decrease in blood wastage in the current study. year 1 of the previous study, showed a decrease in blood wastage by 15.4% (95% ci 13.0–17.7, p ≤ 0.05), year 2 a decrease of 13.1% (95% ci 11.9–14.3, p ≤ 0.05), year 3 a decrease of 12.3% (95% ci 11.2–13.4, p ≤ 0.05). overall, the current study recorded a significant decrease in blood wastage of 12.4% (95% ci 11.6–13.1, p ≤ 0.005). figure 4: percentage of blood wastage recorded in the current study (2016–2018) and in the previous study (2012–2014) 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 year 1 year 2 year 3 total 30.1 26.4 23.4 25.4 14.7 13.3 11.1 13.0 previous study current study seemedj 2021, vol 5, no. 2 blood usage and wastage at a public hospital setting 74 southeastern european medical journal, 2021; 5(2) figure 5: mean ± sd of kap and ag of health personnel regarding blood transfusion (sd = standard deviations; kap = knowledge, attitude and practices; ag = administrative guidelines) kap scores of medical personnel the study included 30 doctors, 31 nurses and 12 medical technologists. the mean (± sd) age of doctors, nurses and medical technologists was 28.9 ± 5.1, 28.5 ± 4.9 and 26.3 ± 4.0, respectively, and the average number of years of experience was 2.9, 4.9 and 4.0, respectively. only the group of doctors included male (30%) and female (70%) participants, while all nurses and medical technologists were female. the mean ± sd (95% ci) scores of kap-ag among doctors, nurses and medical technologists were 27.7 ± 5.1 (95% ci 25.5–29.9), 17.3 ± 3.8 (95% ci 15.7–18.9), 8.7 ± 1.9 (95% ci 7.7–9.7), 2.4 ± 1.3 (95% ci 2.0–2.8); 27.4 ± 6.8 (95% ci 25.2–29.5), 13.0 ± 4.8 (95% ci 11.4–14.5), 7.2 ± 2.7 (95% ci 6.3–8.2), 2.0 ± 1.0 (95% 1.6–2.5); 22.7 ± 6.8 (95% ci 19.2–26.2), 13.0 ± 4.8 (95% ci 11.5–16.5), 9.1 ± 3.7 (95% ci 7.6–10.6) (figure 5). a significant difference was observed among health professionals in regard to knowledge (f = 3.2, p < 0.05), attitude (f = 7.6, p < 0.001) and practices (f = 3.5, p < 0.05); however, no significant difference was observed in regard to administrative guidelines (t = 1.3, p > 0.05). no significant difference was observed between the genders in terms of knowledge (t = -0.5, p > 0.05). discussion this study aimed to investigate the pattern of usage and wastage of blood products before and after simple interventions. likewise, this study aimed to assess the kap of the health personnel regarding blood transfusion. this study was a follow-up study of the study on blood usage and wastage done in a public hospital in guyana in 2012–2014 (9). the previous study indicated a wastage of 25% of blood units (9). to prevent such wastage, several initiatives were introduced in the public hospital under the supervision of the laboratory director, keeping on par with the findings of the previous study. some of the initiatives included increased awareness among hospital staff, encouraging them to maintain the temperature of blood units and to return them before expiry if not used. further, to control expiry of blood products, component identification modalities were included in an electronic registry in order to accurately review and monitor the release of blood units according to the expiry date. a similar study done by collins et al. demonstrated that relatively inexpensive interventions can have a prompt and dramatic impact on reducing blood wastage with regard to both cost and resource savings (14). 27.7 17.3 8.7 2.4 27.4 13.0 7.2 2.0 22.7 14.0 9.1 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 knowledge attitude practice administrative guidelines doctors nurses med tech seemedj 2021, vol 5, no. 2 blood usage and wastage at a public hospital setting 75 southeastern european medical journal, 2021; 5(2) the present follow-up study showed an increase in blood and blood products collection when compared to the previous study done in the same setting (9). this could be assumed to be the result of meeting the demands of the referral hospital and improved management. similar to the previous study, the current study also showed a higher collection of prbc, followed by ffp and pc (9, 15). however, the previous study showed a 25% blood unit wastage, which was greatly reduced to 12.3% blood unit wastage in the present study. the most common causes of blood wastage were expired unit, broken bag, broken cold chain, returning after 30 minutes. similarly, wastage of blood units has also been reported in other studies. in developing countries like iran, blood product wastage of about 9.8% in the qazvin province and of 12% in ahwaz hospitals was reported. blood wastage in developed countries was also reported; 4.4% of issued rbc was wasted according to a study conducted in the united states (us), while a similar study in the uk reported 2.1% to 4.8% blood wastage (13, 16). expiry of blood products was the main cause of wastage in our both current study and in the previous study (9). expiry of blood products also remains the main reason for blood wastage in other studies (3). different intervention strategies have been introduced in various hospitals to reduce blood wastage. educational interventions and simple review and documentation of blood products were introduced after the previous study. this study also included a kap study to understand the basic knowledge, attitude and practices of the health personnel. nurses had good knowledge, attitude and administrative skills concerning blood transfusion, while doctors had good practice scores. the kap study results were not satisfactory and suggested a lack of knowledge and training among doctors, nurses and laboratory technologists regarding transfusion medicine. the results indicated a big gap in the health personnel’s knowledge about transfusion medicine. interns indicated that there was a lack of dedicated teaching or training in the undergraduate curriculum. however, an effective training program for all healthcare personnel would be an essential step in creating good knowledge and effective transfusion practice in any hospital setting. every hospital should work on formulating a hospital transfusion policy and establishing a transfusion committee. this committee should be responsible for providing training to health personnel, implementing policies on blood usage and management of transfusion reactions. such policies hope to reduce unnecessary wastage of blood, as well as provide good knowledge and awareness regarding blood transfusion. conclusion this study showed a decrease in blood wastage from 25% to 13.0% since the introduction of interventions in the public hospital laboratory, guyana. this study therefore represents a model for reducing blood wastage in hospitals worldwide. prevention of blood wastage in hospitals can be achieved with simple, easy and inexpensive interventions, as well as with an efficient inventory system. awareness and training of all healthcare personnel should be encouraged for the purpose of effective blood usage and management in hospitals. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare references 1. world health organization. global forum for blood safety: patient blood management.; 2011. http://www.who.int/bloodsafety/events/gfbs_01_p bm_concept_paper.pdf, 2011 2. world health organization. the 2016 global status report on blood safety and availability 2016.; seemedj 2021, vol 5, no. 2 blood usage and wastage at a public hospital setting 76 southeastern european medical journal, 2021; 5(2) 2017. https://apps.who.int/iris/handle/10665/254987 3. kafi-abad sa, omidkhoda a, pourfatollah aa. analysis of hospital blood components wastage in iran (2005-2015). transfus apher sci off j world apher assoc off j eur soc haemapheresis. 2019; 58(1):34-38. doi:10.1016/j.transci.2018.11.001 4. u.s. food and drug administration. cfr title 21—part 640 additional standards for human blood and blood products, subpart b—red blood cells sec 640.11— general requirements.; 2016. 5. reference standard 5.1.8a: requirements for storage, transportation and expiration. standards for blood banks and transfusion services. 30th ed. bethesda (md): american association of blood banks; 2016:51. 6. british committee for standards in haematology. guideline on the administration of blood components, 2009. http://www.bcshguidelines.com/document s/admin_blood_components_bcsh_050120 10.pdf 7. uk blood transfusion services. tansfusion procedures. in: handbook of transfusion medicine. transfusion procedures. 4th ed. no.; 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30(5):250-255. 13. heitmiller es, hill rb, marshall ce, et al. blood wastage reduction using lean sigma methodology. transfusion. 2010;50(9):1887–96. doi: 10.1097/eja.0b013e32835fadcf. 14. collins ra, wisniewski mk, waters jh, triulzi dj, yazer mh. effectiveness of multiple initiatives to reduce blood component wastage. am j clin pathol. 2015;143(3):329–35. doi: 10.1309/ajcp42wmhsstphxi. 15. sharma a, nallasamy k, jain a, williams v, jayashree m. blood component utilization before and after implementation of good transfusion practice measures in a pediatric emergency department. transfus apher sci. 2020; 59(2):102719. doi:10.1016/j.transci.2020.102719 16. macrate ee, taylor c. on behalf of the blood stocks management scheme (bsms) steering group. the 2011-2012 annual bsms report. london: engl natl heal serv blood stock manag scheme. published online 2012.. 1 author contribution. rk, aa, rb, sa, cc, pr contributed equally to this paper. rk did the statistical analysis and the first draft of the paper. rk and aa contributed to the conception of the study. all of the authors read and approved the final manuscript. seemedj 2022, vol 6, no 1 hypertension, anxiety and depression 31 southeastern european medical journal, 2022; 6(1) review article hypertension in association with anxiety and depression – a narrative review 1 josipa ivanušić pejić ¹, dunja degmečić ¹, ²* 1 department of psychiatry, university hospital centre osijek, croatia 2 faculty of medicine, josip juraj strossmayer university of osijek, croatia *corresponding author: dunja degmečić, ddegmecic@gmail.com received: sep 28, 2021; revised version accepted: feb 20, 2022; published: apr 27, 2022 keywords: blood pressure, hypertension, anxiety, depression, antidepressants, adverse effects abstract hypertension is recognized as a multifactorial disorder. anxiety disorders, depressive disorder, psychosocial stress and certain individual psychological characteristics can influence the development and course of hypertension. likewise, certain antidepressants can impact blood pressure. association of anxiety disorders and depression with hypertension is bidirectional, so hypertensive patients are at risk of anxiety or depression. monitoring the blood pressure of patients with anxiety disorders and depression, screening for anxiety and depression in patients with arterial hypertension and understanding pathophysiological mechanisms is important for future prevention and treatment strategies. this narrative review will briefly summarize current knowledge about the association of anxiety and depression with the risk of development of hypertension. likewise, certain psychological factors and pathophysiological mechanisms in stress that are of importance for the association of hypertension with anxiety and depression are pointed out in this review, and effects of commonly used antidepressants are also considered. (ivanušić pejić j, degmečić d. hypertension in association with anxiety and depression – a narrative review . seemedj 2022; 6(1); 31-43) seemedj 2022, vol 6, no 1 hypertension, anxiety and depression 32 southeastern european medical journal, 2022; 6(1) introduction hypertension is a common chronic disorder in the general population and is itself an important risk factor for cardiovascular disease (1). according to who reports, in 2015, about ¼ of the world’s adult population suffered from hypertension and about 40% of cardiovascular deaths were connected to hypertension (2). blacher et al. showed that each increase of blood pressure by 10 mmhg raises the risk of severe cardiovascular disease complications and death by almost 20% (2). multifactorial aetiology of hypertension includes genetic predisposition as an unmodifiable factor and several potential modifiable factors. the risk of development of hypertension increases with age, especially in women, who are under lower risk in the reproductive period than men. the most important risk factors are smoking, sedentary lifestyle, excessive salt intake and high-calorie food intake, adiposity, as well as stress, anxiety and depression (3). although the association of hypertension with stress, anxiety and depression has been studied for decades, study data are controversial and the underlying pathophysiological mechanisms have not been completely understood. however, it is known that hypertension is understood as a psychosomatic disorder, where psychological factors can play an important role in its development and can have an impact on its course and treatment. there is an impact of psychological factors, psychosocial stressors and mental disorders on the cardiovascular system (4). type a personality, emotional distress, anxiety and depression have the greatest impact on blood pressure (4). recommendations for management of hypertension published in 2018 by the european society of cardiology/european society of hypertension (esc/esh) include psychosocial factors as risk factors for hypertension, while the british national institute for health and clinical excellence (nice) recommended that management of hypertension in adults in primary healthcare settings include interventions for reducing stress and achieving relaxation (5). the aim of this review is to summarize current knowledge about the association of anxiety and depression with the risk of development of hypertension. likewise, certain psychological factors and pathophysiological mechanisms in stress that are of importance for the association of hypertension with anxiety and depression are pointed out in this review. effects of commonly used antidepressants are also considered. methods in order to summarize current knowledge about the association of anxiety and depression with the development of hypertension, we searched for relevant literature using pubmed, sciencedirect, springerlink, psycnet and elsevier until february 2022. keywords included were blood pressure, hypertension, anxiety, depression and stress. blood pressure control the physiological process of blood pressure control is complex and involves regulation of volume and natriuresis, as proposed by guyton, with rapid control (within seconds or minutes) of vessel resistance by the central nervous system (cns) and the sympathetic nervous system (sns), with circadian control (within hours) mostly dependent on activity of the reninangiotensin-aldosterone-system (raas) and with long-term control (within days) mostly reflecting salt intake and activity of raas (6). antihypertensive drugs act through sodium/volume regulation, renin-angiotensin system and the sympathetic nervous system. antihypertensive drugs that act through sodium/volume regulation (diuretics and calcium-channels blockers) or through the renin-angiotensin system (angiotensin converting-enzyme inhibitors and angiotensin receptor blockers) manage to control seemedj 2022, vol 6, no 1 hypertension, anxiety and depression 33 southeastern european medical journal, 2022; 6(1) hypertension in at least 75% or more cases (7). 76% of adult patients diagnosed with hypertension in the usa have suboptimally regulated blood pressure (bp) despite appropriate medication and changes in lifestyle (8). in a minority of hypertensive patients, drugs that act through sns manage to control hypertension better than the drugs mentioned above, so the sympathetic nervous system seems to have a key role (known as neurogenic hypertension in earlier literature) (7). severe hypertension of non-secondary origin, treatment-resistant hypertension (failure of drug combinations that affect sodium/volume and raas) and paroxysmal hypertension (unprovoked severe bp elevation in patients without pheochromocytoma) are considered to be associated with psychological factors (7). certain studies revealed high levels of anxiety among patients with treatment-resistant hypertension (8). paroxysmal episodes of high bp have been observed in patients with unusually severe trauma who deny any emotions and in patients that minimize emotional distress (7). paroxysmal hypertension is often managed acutely with anxiolytics and/or alfa/beta-blockers, and these drugs are also effective in long-term treatment by reducing the magnitude of bp elevation attacks (7). likewise, antidepressants are shown to be successful in preventing paroxysmal bp elevation (7). patients who complain of frequent variations in bp, with particularly high levels accompanied by rapid heartbeat, may also benefit from sns-targeting medications (7). in the 1930s, franz alexander considered the importance of psychological variables in hypertension and he pointed out the relationship of hypertension with repressed hostility (9). expressing anger was shown to be inversely related to blood pressure (9). likewise, reporting less emotional distress and less anxiety negatively affects the blood pressure (9). individuals who are defensive also tend to have higher blood pressure (7). psychosocial stress is thought to be involved in the development of hypertension (10, 11). the risk of developing hypertension is associated with chronic stress, where the intensity of stress is more important than the specific type of stress (10). higher levels of perceived chronic stress, rather than low intensity and short-term stress, are more likely to be accompanied by psychological and behavioural changes that increase the risk of hypertension (10). stronger effects of chronic stress on blood pressure and incident hypertension are seen in women than in men (10). these effects are possibly linked to women being more exposed to psychosocial stressors and having more intense and prolonged emotional and physiological reactions (10). in the jackson heart study, a community-based cohort of black people, moderate and high perceived stress over time were associated with a significantly higher risk (15% and 22%, respectively) of incident hypertension over a median of seven years, compared to sustained low perceived stress (10). consistent results were demonstrated in the coronary artery risk development in young adults study (cardia). the cardia study among black and white participants showed that increasing or sustained levels of stress are associated with incident hypertension among young adults (10). therefore, evaluating prolonged and repeated stress over time can be an important part of primary prevention of hypertension and subsequent risk of cardiovascular disease (10). psychological stressors, according to lazarus, are defined as perceived threats to a person’s well-being that tax or exceed own coping capacity (12). individual differences in coping resources are based on appraisal processes in forebrain neural circuits, where information is assessed in terms of personal relevance and potential threat, after which peripheral physiological reactions are initiated (12). the main response to stressors are immediate changes in the sympathetic and parasympathetic nervous system and hypothalamic-pituitary-adrenal axis, which then influence cardiac output and peripheral vascular resistance to redirect blood flow in peripheral tissue according to behavioural needs (12). cortical and subcortical circuits, according to appraised stressors, generate anticipatory visceromotor commands to change seemedj 2022, vol 6, no 1 hypertension, anxiety and depression 34 southeastern european medical journal, 2022; 6(1) cardiovascular reactions in order to prepare individuals for a behavioural response, and those reactions can be exaggerated in the form of excessive increase of heart rate or blood pressure (12). such exaggerated cardiovascular reactions, when repeated, can have cumulative consequences for cardiovascular disease and cardiovascular incidents among vulnerable individuals (12). brain-imaging studies show that individuals with excessive increase of heart rate and blood pressure in stressful situations exhibit higher activity in the anterior cingulate cortex, medial prefrontal cortex, insula, hippocampus, basal ganglia, periaqueductal grey matter, pons and amygdala (12). short-term psychological stressors evoke cardiovascular reactions which are adaptive, providing hemodynamic and metabolic support for surviving behaviour (known as the fight or flight reaction) (12). however, cardiovascular reactions that are evoked by stressors for a long period of time or in a repeated manner initiate or exacerbate pathophysiological changes in the heart and vasculature (12). blood pressure is controlled by baroreceptors mainly distributed in the bulbus of the carotid artery and the aortic arch (12). increase of blood pressure causes stretching of the baroreceptors, followed by transmission of information through vagal and glossopharyngeal pathways toward nucleus tractus solitarius in the brainstem, which makes neural connections to pre-autonomic nuclei in the brainstem and to the forebrain, such as the ventromedial prefrontal cortex, anterior cingulate cortex, insula and amygdala, which are key regions in threat appraisal (12). these pathways enable modifications in the operating characteristics of the baroreflex through various behaviours, psychological stress, physical activity and particular stages of lifespan and can have a role in disease risk (12). given these findings, it is necessary to study the potential genetic and developmental causes of individual differences in central control of cardiovascular reactions and to prevent psychological reactions and behaviours that increase the risk of cardiovascular disease in response to stress. changes of hypothalamic pituitary adrenal (hpa) axis functioning also underlie stress-induced cardiovascular reactions. chronic psychosocial stress causes continuous secretion of hpa axis hormones, such as cortisol, which lead to resistance of glucocorticoid receptors and consequently reduce sensitivity of the immune system to anti-inflammatory action, thus promoting mild chronic pro-inflammatory state (11). hypertension then develops in the manner that high levels of cortisol inhibit the expression of nitric oxide synthase and thus decrease the availability of endothelial nitric oxide and increase regional vascular resistance (11). chronic low-intensity inflammation also leads to the development of obesity, primarily with intraabdominal accumulation of visceral fat, increased salt retention and insulin resistance (11). increased levels of cortisol and catecholamines in blood, urine and saliva are considered markers of chronic psychosocial stress, but are influenced by physiological fluctuations in circadian rhythms and transient stressors, while hair cortisol concentration is probably a more reliable biological marker reflecting chronic psychosocial stress since cortisol accumulates in the hair for several months (11). in the study by bautista, individuals with high hair cortisol concentrations were twice as likely to be hypertensive compared to those with low levels of cortisol in their hair (11). there exists also an association of alexithymia, which is characterized by an inability to recognize and express emotions, with hypertension (7). hostility, as a personality trait, is a long-term risk factor for hypertension in the general population (cardia study), which also negatively affects the prognosis of coronary heart disease (13). hostile individuals have higher blood pressure and higher rates of cardiovascular morbidity and mortality (13). hostile individuals are cynical and suspicious toward others, their emotions and behaviour cause interpersonal conflicts and lead to a possible lack of social support with perceived fewer coping resources, which can result in difficulties for managing chronic conditions like hypertension and encourage unhealthy habits such as tobacco or alcohol consumption (13). type a personality, characterized by anger, hostility and impatience, seemedj 2022, vol 6, no 1 hypertension, anxiety and depression 35 southeastern european medical journal, 2022; 6(1) increases the risk of coronary heart disease and is a predictor of sudden cardiac death (4). association of anxiety and hypertension anxiety disorders are the most common mental disorders today, with a lifetime risk of occurrence of about 13.5% (2). likewise, according to data of the european study of the epidemiology of mental disorders, the most frequent disorder is the panic disorder – 12.8% will be diagnosed with it (2). anxiety disorders seem to have a central role among all mental disorders associated with hypertension (1). systematic review and meta-analysis of crosssectional and prospective studies by pan et al. supported the association between anxiety and increased risk of hypertension (3). pan et al. conducted a meta-analysis and found significant correlation between anxiety and hypertension in cross-sectional studies and longitudinal association in prospective studies, supporting earlier findings of anxiety as an independent risk factor for incident hypertension (14). in the mechanisms and outcomes of myocardial silent ischemia (momsi) study, incident hypertension in the evaluated period of one year was highly associated with baseline anxiety in middle-aged women, even after adjusting for factors of age, sex, bmi, smoking and psychopharmacotherapy (15). data from world mental health surveys show that of all anxiety disorders, diagnoses of panic disorder, social phobia and specific phobia have been associated with developing hypertension in the period of 11.7 to 34.2 years (15). in the study by wu et al., one year prevalence in 2005 and average annual incidence of hypertension in the period 2006– 2010 in patients with anxiety disorders were higher than in the general population (37.9% vs. 12.4% and 3.63% vs. 1.95%), seen in all age and sex groups (16). in the national health and nutrition examination and epidemiologic follow-up study, in a cohort of initially normotensive men and women that were followed up for 7 to 16 years, anxiety and depression, especially high levels at baseline, were found to be predictive of later experience of hypertension (17). among outpatients with anxiety in the national ambulatory medical care survey and the national hospital ambulatory medical care survey, up to 32.5% had unrecognized elevated blood pressure (18). according to o’donovan et al., individuals that suffer from anxiety disorders show greater threat-related vigilance and sustained threat perception, followed by prolonged activation of the hpa axis and the autonomic nervous system (15). at first, anxiety increases blood pressure transiently; in the long term, it may cause persisting vascular resistance, higher levels of angiotensin ii, sustained sympathetic nervous activity and hpa axis activity as the major stress response system (14). sustained activation of sns reduces renal blood flow, increases sodium and water retention and through abnormal hemodynamic and dysregulating lipid metabolism causes damage and dysfunction of the endothelium, with consequent increase of the risk of atherosclerosis (14). prolonged activation of the neurobiological stress system in different anxiety disorders can potentiate chronic inflammation along with functional and structural damage and, finally, cardiovascular, autoimmune and neurodegenerative diseases can develop (19). anxiety disorders are recognized as associated with higher blood pressure variability (bpv) (15). likewise, the relationship between anxiety and bpv is bidirectional (20). in the study by zhou et al., predictive values of long-term bpv for incident anxiety were studied among patients in family medicine clinics (20). incident anxiety was significantly predicted by higher bp and higher bpv in female and older patients (20). higher bpv is connected to reduced baroreflex sensitivity and it shows sympathetic predominance over decreased parasympathetic activity, which is also found in patients with anxiety disorders (20). higher bpv is a predictor of development, progression and severity of organ damage associated with hypertension (15). small cross-sectional and other studies have shown greater bpv and lower heart rate variability (hrv) among adults with higher levels of anxiety (15). seemedj 2022, vol 6, no 1 hypertension, anxiety and depression 36 southeastern european medical journal, 2022; 6(1) sinhba et al. reported that anxiety levels are negatively correlated with heart rate and hrv in the stress response, and yu et al. found that anxiety may diminish the cardiovascular response to stress by desensitizing betaadrenergic receptors (1). an ambulatory blood pressure monitoring study reported that anxiety disorders were associated with nocturnal and early morning hypertension in hypertensive outpatients (21). numerous studies showed a higher prevalence of panic disorder in hypertensive patients than in the control groups, e.g. davies et al. found that 13% of hypertensive patients in primary care, in contrast with 8% of normotensive patients, suffer from panic disorder, and another study reported that 12% of patients with resistant hypertension have the diagnosis of panic disorder, along with 14% of those without treatment resistance (2). increased lifetime risk of development of hypertension in patients with the diagnosis of panic disorder was reported in prospective studies by grimsrud et al., chou et al. and stein et al. (2). patients with panic disorder show autonomic dysfunction with increased sympathetic and decreased parasympathetic activity, so that increased heart rate and blood pressure and decreased hrv are observed (2). however, some researchers have not found a link between anxiety and hypertension; this was reported in the nord-trøndelag health study (hunt) in norway (8). patients who participated in the hunt study from 1984 to 1986 were reexamined 11 years later and it was reported that high levels of anxiety and depression at baseline predicted low systolic blood pressure at followup (22). researchers also found that increase in symptoms of anxiety and depression from baseline predicted a decrease in blood pressure in men and women of all adult age groups (22). it is important to note that those effects were not explained by the use of antidepressant or antihypertensive medication (22). likewise, patients in the hunt study were re-examined 22 years later and it was found that high levels of combined anxiety and depression at baseline were associated with lower mean systolic and diastolic blood pressure (22). it was reported that the risk of developing hypertension at year 22 was 20% lower and that both anxiety and depression separately contributed to the lowering of blood pressure (22). it is noteworthy that there was no evidence of mediating effects of heart rate changes and that the association of lower blood pressure with anxiety and depression persisted after excluding individuals who used antidepressants or antihypertensive medications (22). it is known that individuals with anxiety disorders, compared to those without such diagnosis, are more likely to smoke, excessively consume alcohol and be obese, but there are studies that show the association of anxiety disorders and prevalent hypertension that is independent of unhealthy behaviour patterns as risk factors for hypertension (15). likewise, patients with anxiety disorders may poorly adhere to medication treatment for hypertension, which makes blood pressure control difficult (11). however, numerous epidemiological studies concerning potential association of anxiety and hypertension have given inconsistent results. in addition to researchers that found that patients with anxiety are at higher risk of developing hypertension, others did not support the role of anxiety in development of hypertension and, finally, a few reported on lower blood pressure in patients with anxiety (14). the australian longitudinal study of women’s health (alswh) reported incident hypertension in 29.8% of women in 15 years of follow-up, but the association was not significant after adjusting for covariates (15). similar findings of generalized anxiety disorder not being significantly correlated with incident hypertension in the elderly after adjusting for covariates were shown in the esther study (15). association of depression and hypertension depressive disorders are the most widespread mental disorders (23). depression is common among patients with cardiovascular diseases, with prevalence estimated between 15% and 50% (4). it is an important risk factor for coronary heart disease and is strongly associated with seemedj 2022, vol 6, no 1 hypertension, anxiety and depression 37 southeastern european medical journal, 2022; 6(1) angina pectoris, myocardial infarction and cardiac death (4). depression leads to greater disability and lower quality of life in cardiovascular patients (23). major depressive disorder is also associated with development of hypertension (1). there are reports of 1.4 times greater chance of having hypertension when depressed (8). the average annual incidence of hypertension in patients diagnosed with major depressive disorder was higher than in the general population (3.96% vs. 2.90%) when studied by liang-wu et al. in the period 2006– 2008, and the 1-year prevalence was also shown to be higher than in the general population (21.21% vs. 13.28%); the results were comparable regardless of sex and age (16). one study conducted on women showed significant increases of systolic blood pressure in relation to increasing depressive symptoms, while increasing intensity of anger and anxiety and decreasing levels of social support showed a greater likelihood of developing hypertension in middle-aged women (24). a meta-analysis by meng et al. showed an increased risk of hypertension incidence in depression, and it was significantly correlated with the baseline intensity of depressive symptoms and length of follow-up (9.6 years) (25). depression is recognized as a significant and independent risk factor for hypertension, especially in younger people, and inducing lower heart rate variability is accompanied by greater mortality of cardiovascular disease (3). in the field of physiological research, a significant correlation has been found between depression, arterial stiffness and hypertension, with the intensity of depressive symptoms independently associated with arterial stiffness (8). there is also growing interest about the role of intracellular calcium homeostasis dysregulation and calcium/camp signalling pathway in the pathogenesis of depression and hypertension (26). calcium channel blockers are known as antihypertensives, but are also reported to have beneficial effects on certain symptoms of depression, such as cognitive dysfunction, and the possible link lies both in regulation of neurotransmitters such as serotonin and sympathetic outflow (26). gonzalez-sanchez et al. reported about increased sympathetic vasomotor tone in patients with depression, association of depressive symptoms with an increase of sns activity and/or decreased pns, cardiac hyperactivity during sns stimulation, morning sbp surge in positive correlation and nocturnal sbp dipping in negative correlation with depressive symptoms (1). the exact mechanism of hypertension development in depression, whether joint with anxiety or separate, still remains incompletely understood (1). the incidence of depression in hypertension patients is 27%, higher than in the general population (13). depression increases the risk of hypertension complications (13). in hypertension patients that also suffer from depression, hypertensive crises are more common than in those without depression (23). villarreal-zegarra showed that the association between hypertension and depression changes as time passes after diagnosing hypertension (27). less than one year after hypertension diagnosis, patients were twice as likely to experience depression compared to individuals without hypertension, and as time passes, the risk of depression in patients with hypertension decreases, but it remains increased even in the period of five years or longer after hypertension is diagnosed (27). possible explanations for this effect can be biological, such as chronic vascular and brain damage under high blood pressure during long periods, and/or psychological, mainly as emotional reactions to awareness of diagnosis and adaption difficulties to personal abilities and need for lifestyle changes. it is probable that patients with arterial hypertension adapt better after a longer period with the diagnosis and thus the risk of depression decreases (27). in the study concerning the impact of anxiety and depression on quality of life among patients with arterial hypertension by polishchuk et al., the majority of hypertensive patients and patients with nonpsychotic mental disorders had high levels of trait and state anxiety, and there was a direct correlation between trait anxiety and severity of depression. levels of anxiety and depression were higher and quality of life was seemedj 2022, vol 6, no 1 hypertension, anxiety and depression 38 southeastern european medical journal, 2022; 6(1) lower in female patients with arterial hypertension, with significantly lower quality of life in patients with mixed anxiety and depressive disorder, as the most common diagnosis in the observed population (28). effects of antidepressants on blood pressure in patients suffering from depressive disorders and anxiety disorders, blood pressure changes have to be considered in the context of taking antidepressants. the longitudinal national study of adolescent to adult health (add health) that followed teenagers to adulthood concluded that taking antidepressants is an independent factor for development of hypertension in men, but not in women, with diastolic bp increased by an average of 1.6 mmhg (8). it has to be questioned whether elevations in blood pressure in patients taking antidepressants are a result of the medication or of the disorder (depression and anxiety disorder) itself. possible effects of antidepressants on blood pressure may be associated with their mechanism of action through serotoninergic, adrenergic, dopaminergic, histaminergic and cholinergic systems (29). selective serotonin reuptake inhibitors (ssris) and serotonin and norepinephrine reuptake inhibitors (snris) may lead to changes of blood pressure through basic mechanisms of inhibiting the serotonin transporter (sert) and the norepinephrine transporter (net). ssris are the most frequently used antidepressants in treatment of depression, but also of anxiety disorders. ssris selectively inhibit sert, but also act through presynaptic serotonin (5-ht) receptor desensitization, mostly 5-ht1a, thus enhancing serotoninergic neurotransmission (29). ssris show limited effects on the autonomic nervous system, which is why they are considered to be a safe class of antidepressants, especially for patients with cardiovascular disorders and older patients; additionally, their long-term use is accompanied by an increase in hr variability, which results in reduced cardiac morbidity and mortality (29). however, humbert et al. analysed the who pharmacovigilance database and showed that the use of ssris significantly increases the risk of hypertension (30). humbert et al. suggested that hypertension can be a possible adverse effect explained by sert inhibition and its vasoconstrictor effects and/or inhibited nitric oxidase vasodilatation effect, while the pharmacoepidemiologicalpharmacodynamic study found a positive correlation of the net/sert pki ratio with the occurrence of hypertension in patients treated with ssris, as well as snris (31). one cohort study in primary care in london was conducted to assess the risk of incident hypertension over 10 years and it showed the correlation of antidepressant use with developing hypertension (and other cardiovascular risks such as dm and hyperlipidaemia), possibly regarding the use of ssris in anxiety treatment (14). citalopram and its enantiomer escitalopram, the most selective ssris, are considered among the safest ssris due to not changing either systolic or diastolic bp nor hr and qtc interval in studies conducted on the older population with or without coronary artery disease (29). sertraline is one of the commonly used ssris and it has the ability to increase dopaminergic neurotransmission. sertraline is also considered one of the safest ssris, even in patients with unstable angina pectoris or after myocardial infarction and among older hypertensive patients (29). fluvoxamine is an ssri with agonistic effect on the sigma-1 receptor. the sigma-1 receptor is believed to mediate vasodilatation and decrease systolic bp in the presence of nitric oxide; however, fluvoxamine is not associated with changes to blood pressure or other significant cardiac effects, although it can cause mild bradycardia and rarely qtc prolongation (29). fluoxetine is an ssri with antagonist effects on 5-ht2c receptors that could enhance noradrenergic and dopaminergic neurotransmission. with short-term use of fluoxetine (12 weeks), depressive patients, either hypertensive or normotensive, show a modest decrease of systolic and diastolic bp, although there are some reports about a larger bp decrease in hypertensive than normotensive seemedj 2022, vol 6, no 1 hypertension, anxiety and depression 39 southeastern european medical journal, 2022; 6(1) patients (29). paroxetine is an ssri that additionally inhibits norepinephrine reuptake and has anticholinergic properties. in patients with ischaemic heart disease and depression, paroxetine shows a significant increase of supine systolic bp after a period of six weeks of administration (29). depressive patients on ssri therapy compared to those on placebo in a meta-analysis by zhong et al. did not show significant changes in blood pressure, while patients on snris compared to patients on ssris experienced a modest increase in systolic and diastolic bp in short-term and long-term treatment (less or more than eight weeks) (32). on the contrary, psychiatric evaluation and ambulatory blood pressure measurements taken in the mayo clinic in florida in the period 2012–2016 showed that patients taking ssris or snris had higher nocturnal systolic and diastolic bp compared to individuals without a diagnosis of mental disorder and without psychopharmacotherapy (33). a single-centre retrospective study among patients with mental disorders who were taking ssris or snris, a group of patients with mental disorders who were not taking ssris or snris and a group of individuals without a mental disorder diagnosis who were not taking psychopharmacotherapy used information obtained from 24-hour ambulatory blood pressure monitoring and showed that ssris and snris were significantly associated with higher nocturnal systolic and diastolic bp (34). that finding is important because nocturnal bp is reported to be a value predictor of cardiovascular mortality regardless of sex and age (34). among snris, venlafaxine shows a significantly higher risk of hypertension in a dose of 150 mg per day, but the risk is reduced by its extended release (er) form (31). it is recommended that venlafaxine users be screened and monitored for hypertension, while duloxetine users are advised to monitor their bp if they have a prior diagnosis of hypertension or cvd (35). in a meta-analysis of 17 randomized clinical trials, park et al. showed that duloxetine does not increase hr and bp in short-term therapy (20). other concerns regarding duloxetine and venlafaxine include their effects on hr and qtc interval because of their described effects on sodium or potassium channels (35), but snris are generally considered safe. tricyclic antidepressants (tcas), however, are connected with serious arrhythmias; they are considered more toxic for the cardiovascular system and should not be administered to cardiac patients (30). tcas inhibit 5-ht reuptake (mainly amitriptyline, imipramine, clomipramine) and ne reuptake (mainly nortriptyline and desipramine), but are also antagonists on cholinergic m1 receptors, histaminergic h1 and adrenergic α1 receptors, and orthostatic hypotension is one of the common side effects. randomized trials have shown that ssris and snris have better safety profiles than tcas, although some meta-analyses reported that citalopram prolonged the qtc interval (36). the use of tcas was accompanied by increased systolic and diastolic bp and patients suffered from hypertension in the study by licht et al., which is why it was concluded that there is an increased risk of hypertension (30). patients with major depressive disorder had a significantly lower systolic bp and anxious patients had a significantly higher diastolic bp compared to healthy controls, while for patients taking tcas, systolic and diastolic bp were significantly higher (37). mirtazapine, a noradrenergic and specific serotoninergic antidepressant (nassa), seems to have a significantly lower risk of adverse cardiovascular effects (36). mirtazapine has antagonistic properties on adrenergic alpha2autoreceptors and alpha2-heteroreceptors as well as 5-ht2 and 5-ht3 receptors, thus enhancing the release of norepinephrine and 5ht1a-mediated serotonergic transmission. in a meta-analysis of 25 randomized and controlled trials by wanabe at al., significantly lower risk of hypertension was shown for mirtazapine than for tcas (30). trazodone is an antidepressant with partial agonistic activity at 5-ht1a receptor and antagonism at 5-ht2a, 5-ht2c, α1 and h1 receptors. trazodone is described as safe for the cardiovascular system, although mild seemedj 2022, vol 6, no 1 hypertension, anxiety and depression 40 southeastern european medical journal, 2022; 6(1) orthostatic hypotension can occur, so the general recommendation is to take blood pressure before administration and to avoid getting up abruptly from a lying or seated position (38). vortioxetine is an antidepressant with multimodal activity; it has antagonistic properties on 5-ht3, 5-ht7 and 5-ht1d receptors, a partial agonistic effect on 5-ht1b and an agonistic effect on 5-ht1a receptors, as well as an inhibitory effect on sert. according to baldwin et al., vortioxetine has a favourable cardiovascular safety profile when compared to the placebo in acute randomized controlled trials (six to eight weeks in duration) and in longterm open-label treatment (52 weeks of duration) (39). agomelatine is an antidepressant with agonistic properties at melatonin (mt1 and mt2) receptors and antagonistic properties at 5-ht2b/5-ht2c receptors. in addition to the effect on the circadian rhythm, agomelatine can influence the autonomic output to the cardiovascular system; however, agomelatine seems to have a safe cardiovascular profile (29). clinical studies suggest agomelatine as a safe option among antidepressants in patients with cardiovascular disease; additionally, its cardioprotective and anti-inflammatory effects are described (40). moclobemide is a reversible inhibitor of monoaminoxidase (maoi) and through that mechanism, breakdown of monoamines is reduced. earlier maois were irreversible and had a greater risk of increasing blood pressure, even causing a hypertensive crisis, when taken together with tyramine-rich food and medications like phenylephrine and pseudoephedrine. the general recommendation is to avoid such food and medications when taking maois (30). further studies for the assessment of effects of commonly used antidepressant medications are needed – particularly an analysis of potential association of changes in blood pressure to the prescribed doses of antidepressants. conclusion aetiology of hypertension has been widely studied in the last few decades. it is known that hypertension is the result of complex interaction of genetic and environmental risk factors, especially lifestyle and psychosocial factors. mental disorders, emotional distress and certain individual psychological characteristics have been associated with increased incidence of hypertension. anxiety disorders and depression have been established as risk factors for hypertension. as hypertension is itself a major preventable factor for cardiovascular disease, cerebrovascular diseases and chronic kidney damage and is associated with earlier mortality, better understanding of the pathophysiological mechanisms underlying the association of anxiety and depression with hypertension could improve prevention and treatment of hypertension. implementing systematic screening for hypertension among patients suffering from anxiety disorders or depression can be questioned. because of the bidirectional relationship and possible negative effects of anxiety disorders and depression on hypertension, treatment and quality of life, patients should be screened for mental disorders and, if needed, referred to clinical psychological interventions and administered psychiatric medication, i.e. antidepressant therapy. knowledge about the effects of antidepressants on blood pressure is of importance and enables the optimal selection of medications for patients with anxiety disorders or depression, especially those for whom other risks of development of hypertension are present or those that are already hypertensive. acknowledgement. none. disclosure funding. no specific funding was received for this study competing interests. none to declare. seemedj 2022, vol 6, no 1 hypertension, anxiety and depression 41 southeastern european medical journal, 2022; 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30(3):242-252. doi:10.1177/0269881116628440 hong n, ye z, lin y, liu w, xu n, wang y. agomelatine prevents angiotensin ii-induced endothelial and mononuclear cell adhesion. aging (albany ny). 2021; 13(14):18515-18526. doi:10.18632/aging.203299 1 author contribution. acquisition of data: ivanušić pejić j, degmečić d. administrative, technical or logistic support: ivanušić pejić j analysis and interpretation of data: ivanušić pejić j conception and design: ivanušić pejić j, degmečić d. critical revision of the article for important intellectual content: ivanušić pejić j, degmečić d. drafting of the article: ivanušić pejić j. final approval of the article: ivanušić pejić j, degmečić d. seemedj 2017, vol 1, no. 1 functional carotid ultrasound markers of subclinical atherosclerosis in men… 19 southeastern european medical journal, vol 1, 2017. functional carotid ultrasound markers of subclinical atherosclerosis in men with cardiovascular risk factors 1 krunoslav buljan1, mirta benšić2, vesna buljan3, mirjana vladetić1, silva butković soldo1 1 department of neurology, osijek university hospital, faculty of medicine, university josip juraj strossmayer osijek, osijek, croatia 2 department of mathematics, university josip juraj strossmayer osijek, osijek, croatia 3 institute of public health for the osijek-baranya county, osijek, croatia corresponding author: krunoslav buljan, md, phd krunoslavbuljan@net.hr received: march 8, 2017; revised version accepted: april 13, 2017; published: april 24. 2017 keywords: ultrasonography; carotid atherosclerosis; risk factors; arterial stiffness abstract aim: the aim of this study was to compare the most commonly used ultrasound parameters of elasticity and stiffness of the arterial wall in detection of carotid subclinical atherosclerosis (sa) in vascular healthy men with one of the most prominent cardiovascular risk factors (cvrf). methods: total of 120 vascular healthy men between 33 and 59 years of age (�̅�(sd) = 49.7(6.93)) were allocated into hypertension, diabetes, smokers and control group of respondents without cvrf. ultrasound examination of carotid arteries was used to measure intima-media thickness and maximal and minimal lumen diameter. along with the blood pressure of all the respondents, the following markers of elasticity/stiffness of arterial wall were calculated: distensibility coefficient (dc), compliance coefficient (cc), young's elasticity modulus (yem) and β stiffness index (β). results: dc, cc and β indicated significantly lower elasticity and higher stiffness of arterial wall in hypertension and diabetic groups of respondents without cvrf (multiple comparison, p<0.001). there were significant changes in functional characteristics of carotid arteries present between respondents-smokers and the control group detected by dc and β (multiple comparison, p<0.05). there were 94 respondents (78%) with subclinical carotid atherosclerosis criteria. roc analysis indicated that dc, β and cc (auc 0.811, 0.810, 0.799) are good markers for sa. conclusions: in conclusion, it is possible to use an ultrasound in detection of changes of elasticity/stiffness in arterial wall caused by the major cvrf in vascular healthy men. dc and β seem to be the best indicators of the presence/absence of subclinical carotid atherosclerosis. (buljan k, benšić m, buljan v, vladetić m, butković soldo s. functional carotid ultrasound markers of subclinical atherosclerosis in men with cardiovascular risk factors. seemedj 2017;1(1);19-26) seemedj 2017, vol 1, no. 1 functional carotid ultrasound markers of subclinical atherosclerosis in men… 20 southeastern european medical journal, vol 1, 2017. introduction subclinical atherosclerosis (sa) is a latent precursor of cardiovascular disease, but the complete prevalence remains unfamiliar (1). there is a high level of clinical interest in detecting vascular changes by non-invasive methods, as it could contribute to identifying individuals at high risk of cardiovascular incident, who might also be in need of more aggressive approach to risk factors (2). sa indicates the cumulative effect of all known and unknown risk factors together, thus providing better data than the data evaluation of only known risk factors (3). different types of ultrasound tests of subclinical atherosclerosis bear different prognostic features. there was an independent correlation recognized between the changes of the elastic properties of the carotid arteries and cardiovascular outcome (4) as well as ischemic stroke (5). the aim of this study was to compare the most commonly used ultrasound parameters of elasticity and stiffness of the arterial wall in the detection of carotid sa in vascular healthy men with one of the most prominent cardiovascular risk factors (cvrf). patients and methods the research was conducted from october 2011 to june 2014 and it included 120 men from eastern croatia (osijek and 40 km radius) between 33 and 59 years of age (x̅(sd)=49.7 (6.93)), who had never had a vascular disease. the respondents were patients referred to diagnostic procedures or specialist examinations at the clinical hospital center osijek; they were diabetic patients hospitalized at the department of endocrinology at the clinical hospital center osijek and volunteers (who were mostly part of the control group). three risk factor criteria including hypertension, diabetes type ii and smoking were indicators in forming three groups; the risk factors being present over the last five years. the control group consisted of respondents without cvrf. to eliminate the possibility of arterial hypertension (according to the american heart association), each respondent’s blood pressure was set at <140/<90 (6). to exclude the diabetes, fasting blood glucose (fbg) level was set at <7.0 mmol/l and the value of glycated hemoglobin (hba1c) was set at ≤6.5% (according to the american diabetes association) (7). respondents smokers were men who smoked for longer than five years and more than 10 cigarettes a day on average, while non-smokers were identified as men who have not actively been smoking for the last five years, and in time prior to that no more than 10 cigarettes per day in a period of one year (8). excluding factors were the presence of another cvrf, i.e. anamnestic or medical history data of arterial disease, both present or past (i.e. myocardial infarction, angina pectoris, ischemic cardiomyopathy, cerebral infarction, transient cerebral ischemic attack, peripheral arterial disease, abdominal aortic aneurysm and cardiac arrhythmia) or previous therapeutic procedure of arterial disease (i.e. percutaneous transluminal angioplasty, endarterectomy, intraarterial stent implantation, arterial bypass). the carotid arteries ultrasound scan of all respondents has been performed in b-mode by the same examiner (kb), by linear probe 7.5 mhz on aloka pro sound 5000, tokyo, japan. carotid atherosclerotic plaque was defined by mannheimcarotid intima-media thickness and plaqueconsensus (9, 10) and it was observed in the segment including 30 mm distal common carotid arteries (cca), carotid bulb and proximal 20 mm internal carotid arteries on both sides of the neck. intima-media thickness was measured by the standard protocol on the far wall of the distal segment of both cca, 10 mm proximal of the starting point of bifurcation (9), with multiangle insonation (anterolateral, lateral and posterolateral) (11), in diastolic blood flow phase with maximum magnification of the image. the mean of carotid intima-media thickness (cimt) is set for the right and left side for each respondent. ultrasound criteria for defining sa were the presence of atherosclerotic plaque on carotid arteries and/or cimt values ≥75th percentile of the control group (11, 12). the measurement of the minimal diastolic diameter of cca between two lines of adventitia-media was performed 10mm proximal of bifurcation seemedj 2017, vol 1, no. 1 functional carotid ultrasound markers of subclinical atherosclerosis in men… 21 southeastern european medical journal, vol 1, 2017. (13), three times on each side, and the lowest value was taken as the referential one. the maximum systolic and minimum diastolic lumen diameter of common carotid arteries in intimamedia area of close and far arterial wall was measured in maximum systolic expansion of the artery and minimal lumen width, during the relaxation of the artery at the end of diastole (14). measuring was performed four or five times on each artery, with the maximum image magnification, along with the examination of previously recorded and stored images over three to five cardiac cycles (15). the results of the maximum and minimum diameter were an average of two maximal systolic and an average of two minimal diastolic lumen diameters for each respondent. just before the first and during the carotid arteries diameter measurement, the blood pressure was also measured on the upper arm side that corresponded to the test of the current carotid artery. it was taken by automatic electronic sphygmomanometer omron m6 comfort, kyoto, japan, which was validated according to the international protocol of the european society of hypertension (16). the conversion factor of the measured blood pressure from mmhg to kpa is 0.13. ultrasound elasticity markers of the carotid wall distensibility coefficient (dc) and compliance coefficient (cc) were calculated according to the following: 𝐷𝐶 = ( 2∆𝐷 𝐷𝐷 ) ∆𝑃 [𝑘𝑃𝑎−1] (15, 17), 𝐶𝐶 = 𝜋𝐷𝐷 ∆𝐷 2∆𝑃 [𝑚2𝑘𝑃𝑎−1] (17, 18). stiffness markers of arterial wall – young’s elasticity modulus (yem) and beta stiffness index (β) were calculated according to the following: 𝑌𝐸𝑀 = 𝐸𝑃𝐷𝐷 2𝐶𝐼𝑀𝑇 [𝑘𝑃𝑎], where 𝐸𝑃 = ∆𝑃𝐷𝐷 ∆𝐷 [𝑘𝑃𝑎] (18, 19) and 𝐸𝑃 = ∆𝑃𝐷𝐷 ∆𝐷 [𝑘𝑃𝑎] where 𝑆𝑇𝑅𝐴𝐼𝑁 = ∆𝐷 𝐷𝐷 [%] (18,20). in the above expressions, dd is cca lumen diameter at the end of diastole, ∆d is pulsatile diameter change, i.e. the difference in systolic and diastolic diameter of the cca lumen, ∆p is pulse pressure, i.e. the difference in systolic and diastolic pressure readings, ep is peterson's (elastic) modulus, ln is natural logarithm, and strain is the change in lumen diameter of the cca during cardiac cycle (expressed in percentage). total cholesterol was quantitatively measured by enzymatic spectrophotometric method, while hscrp was measured by turbidimetry (beckman coulter au 680 analyzer). blood glucose was measured quantitatively by uv enzymatic test (hexokinase method) on beckman coulter au 680 analyzer. hba1c was quantitatively measured by turbidimetric inhibition immunoassay (tinia) principle on dimension clinical chemistry system rxl, siemens. statistical analysis wilcoxon’s signed-rank test, when applied to 17 respondents, did not indicate statistically significant difference in the first and second (performed within three months) ultrasound parameters of the following: cimt, diameter, ∆d. furthermore, statistically significant difference was not found in the number of subjects with or without carotid atherosclerotic plaque. figure 1. distensibility coefficient of the respondents with the cardiovascular risk factor. seemedj 2017, vol 1, no. 1 functional carotid ultrasound markers of subclinical atherosclerosis in men… 22 southeastern european medical journal, vol 1, 2017. in order to verify the difference in the expected numerical values of arterial walls, depending on their groups, t-test, i.e. anova test was used. in some cases, in order to verify the difference in data distribution, it was necessary to use kruskal-wallis test, mann-whitney u test for independent samples, or wilcoxon's signedrank test for paired samples. the effectiveness of the numerical variable in subclinical atherosclerosis risk assessment is discussed because of its sensitivity and specificity, but also because of the analysis of the roc curve results. significance level was set at 0.05 in all of the tests. statistical analysis was carried out in the data analysis program r (https://cran.rproject.org, package rocr and proc) and statistica (statsoft, version 11, http//www.statsoft.com/company). results table 1 presents basic hemodynamic and metabolic variables of respondents with respect to cvrf. statistically significant difference in age and levels of total cholesterol has not been found between the control group and risk factor groups (p=0.741, i.e. p=0.904). the hypertension group indicated the highest values of blood figure 2. roc curve of functional ultrasound tests of the carotid arteries with subclinical atherosclerosis prediction. table 1. average value (±sd) of hemodynamic and metabolic variables of respondents with respect to cardiovascular risk factor. men (n=120) cardiovascular risk factor control group (n=30) p hypertension (n=28) diabetes (n=30) smoking (n=32) �̅�(sd) age, years 49,7(8,16) 50,9(5,69) 49,1(6,94) 49,1(6,53) 0,741‡ duration of the risk factor, years 8,2(3,72) 10,8(4,32) 26,1(6,79) <0,001§ systolic blood pressure, mmhg 145,1(16,46) 128,8(9,38) 127(10,32) 126(12,18) <0,001§ diastolic blood pressure, mmhg 90,6(10,84) 81,7(5,37) 81,6(7,15) 80,3(8,17) <0,001§ total cholesterol, mmol/l 5,73(0,874) 5,81(2,415) 5,95(1,024) 5,69(0,97) 0,904‡ hscrp*, mg/l 2,012(1,371) 3,018(2,579) 3,52(4,964) 1,096(0,701) <0,001|| fasting plasma glucose, mmol/l 5,25(0,692) 9,99(2,658) 5,24(0,745) 4,96(0,419) <0,001§ hba1c†, % 5,62(0,375) 9,21(2,02) 5,68(0,437) 5,52(0,301) <0,001§ *high-sensitivity c-reactive protein; †glycosylated hemoglobin a1c; ‡classical anova; §welch's anova; ||kruskal-wallis anova https://cran.r-project.org/ https://cran.r-project.org/ file:///c:/users/marija%20r/appdata/local/temp/http/www.statsoft.com/company seemedj 2017, vol 1, no. 1 functional carotid ultrasound markers of subclinical atherosclerosis in men… 23 southeastern european medical journal, vol 1, 2017. pressure, while fbg and hba1c were significantly higher in diabetic respondents (table 1). smokers indicated statistically significant higher values of hscrp when compared to other risk factor groups and control group (p<0.001). ultrasound markers of carotid arteries’ elasticity – dc and cc – indicated differences between the control group and groups of respondents with hypertension and diabetes (multiple comparison, p<0.001), while indicating the difference of smokers in dc, but not in cc (multiple comparison, p=0.033, i.e. p=0.055) (table 2, figure1). the analysis of β stiffness index indicated significantly higher arterial stiffness in respondents belonging to the hypertension and diabetic groups (multiple comparison, p<0.001), as well as smokers (multiple comparison, p=0.028) in comparison with the control group. yem values, as the second arterial stiffness indicator, proved to be the highest in the hypertension group, i.e. they were significantly higher than those in the control group (p<0.001) (table 2). pearson’s correlation coefficient indicated a significant positive correlation of age and β (0.507; p<0.001), and negative correlation of age and dc (-0.496; p<0.001), independent of cvrf presence/absence. roc analysis of functional vascular ultrasound parameters with carotid sa prediction was done with respect to the sa ultrasound criteria – cimt values ≥75th percentile in the control group and/or presence of the atherosclerotic plaque in carotid arteries (figure2, table 3). there were 94 out of 120 respondents (78%) with one or two subclinical carotid atherosclerosis criteria and 26 (22%) without any of the mentioned criteria. of all the functional ultrasound tests in predicting sa, the following seem to be the best indicators: dc (auc 0.811), ß (auc 0.810) and cc (auc 0.799). the borderline value of dc to discriminate between the respondents with and without sa was set at ≥0.0231 kpa-1 (sensitivity 0.872, specificity 0.538), and for ß≤4.47 (sensitivity 0.872, specificity 0.538). roc analysis of the ultrasound tests of carotid arteries has been lower in participants with cvrf (n=90, group+ =78, group=12). according to auc data, dc, cc, yem and ß were lower (0.806, 0.784, 0.513 and 0.798) than in roc analysis, which also included the respondents from the control group. discussion given that numerous research has already reported the correlation of age and arterial elasticity/stiffness indicators (18, 21-23), which has been corroborated in our research as well, the absence of significant age difference has been a key prerequisite of this research. as expected, the highest difference in blood pressure values was found in the hypertension table 2. ultrasound carotid arteries’ parameters of elasticity and stiffness with respect to the risk factor. men (n=120) functional ultrasonic parameter cardiovascular risk factor control group (n=30) p¶ hypertension (n=28) diabetes (n=30) smoking (n=32) x̅(sd) dc*, kpa-1 0,0157 (0,0129-0,017) 0,0154 (0,0136-0,0183) 0,0182 (0,0148-0,0231) 0,0251 (0,0173-0,0279) <0,001 cc†, 10-7m2kpa-1 4,81 (4,15-5,56) 4,26 (3,65-5,24) 5,21 (4,7-6,49) 6,46 (5,42-7,27) <0,001 yem‡, kpa 623,2 (553-717,3) 529,9 (428,4-616,6) 533,2 (394,7-615,5) 444,2 (368,6-547,4) <0,001 β§ 4,86 (4,78-5,05) 4,87 (4,72-4,99) 4,71 (4,46-4,92) 4,38 (4,27-4,75) <0,001 *distensibility coefficient; †compliance coefficient; ‡young's elasticity modulus; §beta stiffness index; ||interquartile range; ¶kruskalwallis test seemedj 2017, vol 1, no. 1 functional carotid ultrasound markers of subclinical atherosclerosis in men… 24 southeastern european medical journal, vol 1, 2017. group, while fbg and hba1c were found in respondents with diabetes. hscrp was significantly higher in smokers than in nonsmokers (p<0.001), similar to previous research (24, 25). in this research, dc, cc and ß index indicated statistically significant lower elasticity and higher stiffness of the carotid arterial wall in respondents with diabetes, hypertension and smokers when compared to healthy participants without cvfr. these findings corroborate the results found in previous research (21, 26-28). similar to our results, sharret et al. found higher arterial wall stiffness in respondents with diabetes, but they have also found the correlation between smoking and higher elasticity of the arteries (29). by contrast, our research found ß and dc to be the ultrasound parameters that indicate statistically lower elasticity and higher stiffness of the arterial wall in smokers in comparison with the control group (p<0.05). the previous literature did not account for the comparison of values of different functional ultrasound parameters in detecting subclinical carotid atherosclerosis. roc analysis indicated that dc, ß, and cc seem to be valid predictors of the subclinical carotid atherosclerosis in vascular healthy men, independently of the presence/absence of the cvrf. yem, when compared to other functional ultrasound tests, did not indicate significant differences in determining the presence/absence of the sa of the observed groups of men. automatic measurement of the oscillation of carotid arteries’ lumen width was not performed in this research due to technical malfunction of necessary appliances. according to prado et al., some recent research used standardized manual approach, which could be as precise as automatic measuring (18). cuadrado godia et al. have reported good reproducibility of cca diameters measured with b/m-mode sonography in their results (30). conclusions to conclude, the results of this research indicate the possibility of ultrasound detection of early arteriosclerotic changes in elasticity and stiffness of carotid arteries in vascular asymptomatic hypertensive and diabetic respondents, as well as smokers. dc and ß seem to be the most prominent ultrasound markers, which could differentiate between male respondents with or without sa better than cc and yem. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. toth pp. subclinical atherosclerosis: what it is, what it means and what we can do about it. int j clin prac 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blood cholesterol differ in the associations with subclinical atherosclerosis: the multiethnic study of atherosclerosis (mesa). atherosclerosis 2006;186:441-7.doi: http://dx.doi.org/10.1016/j.atherosclerosis. 2005.08.010. 30. cuadrado godia e, madhok r, pittman j,trocio s, ramas r, cabral d, sacco rl, rundek t. carotid artery distensibility. j ultrasound med 2007;26(9):1157-65. seemedj 2022, vol 6, no 1 iron deficiency and recurrent aphthous stomatitis 105 southeastern european medical journal, 2022; 6(1) review article the correlation between iron deficiency and recurrent aphthous stomatitis: a literature review 1 antonija mišković 1,2*, nikica marinić 2,3, zvonimir bosnić 2,4, karolina veselski 1,2, domagoj vučić 2,5, ivana pajić matić 1,6 1 ent department, general hospital “dr josip benčević”, slavonski brod, croatia 2 university postgraduate interdisciplinary study – molecular biosciences, josip juraj strossmayer university of osijek, osijek, croatia 3 private dental medicine practice nikica marinić, slavonski brod, croatia 4 family medicine practice zvonimir bosnić, slavonski brod, croatia 5 department of cardiology, general hospital “dr josip benčević”, slavonski brod, croatia 6 faculty of medicine, josip juraj strossmayer university of osijek, osijek, croatia *corresponding author: antonija mišković, miskovic.antonija@gmail.com received: dec 12, 2021; revised version accepted: feb 27, 2022; published: apr 27, 2022 keywords: iron, aphthous stomatitis, iron deficiency anemia abstract aphthous lesions of the oral mucosa are a very common symptom and can be seen in both family medicine practice, dental medicine practice, and dermatology or otorhinolaryngology clinics. some patients develop a chronic recurrent condition, which is clinically known as recurrent aphthous stomatitis (ras). these ulcers are round, clearly defined, and can be visible on the movable part of the oral mucosa, with variations in size. a prodromal symptom like the burning or stinging sensation can precede the appearance of lesions. the main reason why patients seek medical help is oropharyngeal pain with lack of appetite. the exact etiopathogenesis of ras remains unknown. immune disorders, nutritional deficiencies, allergies, mechanical injuries, and even psychological disorders are being studied as potential causes of this condition. some authors claim that iron deficiency may be a possible causative factor of ras due to its role in dna synthesis, mitochondrial function, and enzymatic activity. in iron deficiency, epithelial cells turn over more rapidly and produce an immature or atrophic mucosa. such mucosa is vulnerable and can be a fertile soil for chronic inflammation and development of aphthae. finally, our goals were to describe the clinical aspects and etiology of ras, as well as to determine whether ras may be related to iron deficiency, in order to identify potential patients with iron deficiency in everyday work. (mišković a, marinić n, bosnić z, veselski k, vučić d, pajić matić i. the correlation between iron deficiency and recurrent aphthous stomatitis: a literature review. seemedj 2022; 6(1); 105-112) seemedj 2022, vol 6, no 1 iron deficiency and recurrent aphthous stomatitis 106 southeastern european medical journal, 2022; 6(1) introduction recurrent aphthous stomatitis (ras) is a frequent oral disorder characterized by multiple painful ulcers in the oral cavity and oropharynx. the prevalence of this condition in the general population is high and varies from 5% to 66%, with a mean of 20% (1). it can be seen in both family medicine practice, dental medicine practice, and dermatology or otorhinolaryngology clinics. we therefore wanted to describe the clinical aspects and etiology of ras, as well as determine whether ras may be related to iron deficiency, in order to identify potential patients with iron deficiency in everyday work. methods of literature search we performed a literature search using pubmed and google scholar and we used the search filter with the following specific terms: recurrent aphthous stomatitis, aphthae, iron deficiency, and anemia, to find works published in the last twenty years, from 2000 until now. in searching these databases, we focused on meta-analyses, systematic reviews, randomized controlled trials, and landmark studies that have previously focused on similar topics (table 1). table 1. results of international research on hematinic deficiency in ras c = control group, ras = recurrent aphthous stomatitis, m = men, w = women, x = no data. classification of recurrent aphthous stomatitis the onset of ras is usually during childhood, with symptoms decreasing with age. the ulcers are round or oval and very painful. when looking closely at each aphtha, an erythematous lesion with a raised edge and inner necrosis covered with a yellow pseudomembrane can be seen. a author and year of study country no. of subjects serum iron level (µg/dl) ferritin (ng/ml) hemoglobi n (g/dl) anemia (n) all m w m w m w slebioda et al. 2018 poland ras 71 141 104.96 78.56 x x 7 c 70 115.85 102.43 x x 1 sun et al. 2014 taiwan ras 273 546 96.8 83.9 x 14.1 12.8 57 c 273 104.1 97.9 x 15.1 13.6 0 piskin et al. 2001 turkey ras 35 61 91.39 71.06 63.78 53.47 x x c 26 84.23 75.31 59.74 67.73 x x al-amad et al. 2019 united arab emirates ras 52 104 81.0 x 14.0 13 c 52 89.3 x 14.5 9 koybasi et al. 2006 turkey ras 34 64 67.82 x 13.53 x c 32 71.16 x 13.42 x babaee et al. 2015 iran ras 28 56 x 115.64 12.87 x c 28 x 55.42 12.98 x low, number low, number lopezjornet et al. 2013 spain ras 92 186 low (< 60 µg/dl) = 7 low (< 12 ng/ml) = 6 x x c 94 low (< 60 µg/dl) = 2 low (< 12 ng/ml) = 5 x x compilato et al. 2010 italy ras 32 61 low (< 40 µg/dl) = 11 low (< 10 ng/ml) = 13 x 11 c 29 low (< 40 µg/dl) = 2 low (< 10 ng/ml) = 0 x 2 seemedj 2022, vol 6, no 1 iron deficiency and recurrent aphthous stomatitis 107 southeastern european medical journal, 2022; 6(1) burning or stinging sensation can precede the appearance of erythematous macules and consequently very painful ulcers are formed (2). clinically, ras can be classified into three types: minor, major, and herpetiform. small aphthous ulcers (minor ras), which are smaller than 10 mm in diameter, appear in more than 80% of patients and heal without a scar in 7 to 14 days. the most commonly affected sites are buccal and labial mucosa. large aphthous ulcers (major ras), which are bigger than 10 mm, heal in 2 to 6 weeks and often leave scars (3). the soft palate, tonsillar fossa, and labial mucosa are frequently affected. herpetiform ras is rare and represents a condition in which a great number of small oral lesions is seen. the typical onset of herpetiform ras is in adulthood and it resembles herpes simplex virus type 1 infection, which is usually seen on the gingiva and hard palate (1). etiology of recurrent aphthous stomatitis recurrent aphthous stomatitis is one of the recurrent ulcerative disorders of the oral cavity. the exact causative mechanism remains unclear, but experts agree on its multifactorial origin (4). possible etiologic factors of ras include dysregulation of the immune system, hematologic diseases, hypovitaminosis and mineral deficiencies, allergies, mechanical injuries, mental disorders, etc (5) (figure 1). figure 1. etiology of recurrent aphthous stomatitis some studies suggest that local trauma may be the main cause of aphthae development, but only in susceptible individuals with a hereditary predisposition to the disease (6). genetic predisposition plays a significant role in the development of ras. the eruption of aphthae is more common in patients with positive family history. likewise, it is followed by a severe clinical presentation (4). guimaraes et al. examined whether dna polymorphism related to the cytokine interleukin-1 beta (il-1 β) may be associated with ras. their work showed a positive correlation and demonstrated how a genetic risk factor can determine susceptibility to the disease. aberrant cytokine cascade induces an amplified cell-mediated immune response and activation of t-lymphocytes and phagocytes. these immune cells are directed seemedj 2022, vol 6, no 1 iron deficiency and recurrent aphthous stomatitis 108 southeastern european medical journal, 2022; 6(1) toward focal areas of the oral mucosa, resulting in ulceration (7). iron in recurrent aphthous stomatitis in some patients, the possible causative factors are vitamin and mineral insufficiency, such as lack of serum iron, folate, zinc, or vitamin b12, and their frequency is estimated at 5%–15% (8). iron is a very important element in the human body. hemoglobin, myoglobin, and a variety of enzymes contain the majority of the iron. the main storage of iron within the human body is the liver, where it is stored in the form of ferritin and hemosiderin. transport of iron through the plasma is performed by the transport protein transferrin, which binds to its receptor when it reaches the tissue (9). when looking intracellularly, iron is essential for dna replication, enzyme and mitochondrial action, and neurotransmitter function (10). when iron intake is too low to fulfill normal bodily demands or to satisfy a pathological deficit, stored iron is consumed, leading to a lack of available iron for hematopoiesis and erythropoiesis. production of hemoglobin without adequate concentrations of iron culminates in hypochromic microcytic anemia (9). moreover, a lack of iron will disrupt lymphocyte development and impair cytokine production, leading to decreased cellular immunity (11). cytochrome oxidase, the iron-dependent enzyme, is required for the development of epithelial cells (12). in iron deficiency, epithelial cells turn over more rapidly and produce an immature or atrophic mucosa (13). such impaired mucosa is more sensitive and susceptible to a pathological process like ulcer development. in addition, lower oxygen levels in anemic patients mean lower oxygen levels in different tissue, as well as the mucosal epithelium of the oropharynx. inadequate oxygen saturation contributes to the disintegration of cells, which provokes atrophic mucosa. atrophic oral epithelium may be the cause of increased susceptibility to ras development in anemic patients (14). the influence of age-related changes on the immune system and development of recurrent aphthous stomatitis aging is related to a higher level of systemic inflammation and persistent activation of nonspecific immune cells due to lifelong exposure to external antigens and raised stimulation with own antigens (15). disbalance in the inflammatory response leads to a variety of common chronic diseases due to dysregulation of immune cells and lack of their self-limiting feature, although in the elderly, the driving mechanism is more complicated, considering the vast number of comorbidities (16, 17). unfortunately, current understanding of the relationship between the physiological processes in aging and the development of agerelated illnesses remains inconclusive, with a lack of standardized methodology to clinically evaluate the inflammation. assessment of iron status is challenging when concomitant inflammation is present. identifying how to optimally adjust conditions linked with chronic illnesses and how to select realistic target outcomes at various stages of disease progression is becoming increasingly difficult (18). furthermore, difficulties in stress-induced physiological mechanisms result in a detrimental allostatic load, a cumulative burden of chronic stress and life events (20), eventually precipitating a negative stress response along with disease progression, especially in chronic medical conditions (19). due to its association with endothelial dysfunction and chronic inflammation, it can be assumed that the frequency of ras would be higher in the elderly, but this would be incorrect. the occurrence of ras decreases with age, while its severity increases (21). however, there is no clear understanding of the pathophysiological mechanism. the first clinical presentation of aphthous lesions in the elderly can easily be a local sign of systemic disease, which is why the term “aphthous-like ulcers” is used (22). seemedj 2022, vol 6, no 1 iron deficiency and recurrent aphthous stomatitis 109 southeastern european medical journal, 2022; 6(1) discussion the effect of iron deficiency in the biology of ras has been studied by various authors with inconclusive results (table 1). koybasi et al. (23) examined the possible etiologic factors of ras in a controlled prospective study by analyzing 34 patients with recurrent aphthous stomatitis and 32 healthy subjects. they identified some important predisposing factors, such as positive family history and vitamin b12 deficiency. interestingly, their study showed that nonsmoking status is connected with a higher chance of aphthae development, which means that smoking can have a protective effect. this can be explained by increased keratinization of the oral cavity due to cigarette use. finally, their results did not show a statistically significant correlation between iron, ferritin, hemoglobin and ras. piskin et al. (24) studied 35 patients with ras and 26 control subjects. they found that patients with oral ulcers have iron, ferritin, and folic acid insufficiency more frequently, but that this is statistically irrelevant (p > 0.05). vitamin b12 level was the only measured variable that showed significance (p = 0.005). al-amad and hasan (25) conducted a casecontrol study on 52 patients with stomatitis and 52 age-matched healthy subjects. their results showed a high percentage of deficiencies in the overall study population. low hemoglobin was seen in 22%, vitamin b12 deficiency in 15%, iron deficiency in 11%, and vitamin d deficiency surprisingly in 53% of the subjects, with no difference between the diseased and healthy cases. they pointed out that vitamin d deficiency makes ulcers more severe. lopezjornet et al. (26) evaluated laboratory tests of 92 patients with ras and 94 healthy controls. they measured the concentration of iron, ferritin, folic acid, and vitamin b12. their results showed that the patients with ras have hematinic deficiencies more often compared with healthy controls. however, this result was also statistically insignificant, with overall frequency of hematinic deficiencies in the ras group amounting to 14.14% and in the control group to 6.39% (p = 0.086). compilato et al. (27) studied 32 adults with ras and 29 healthy controls, measuring full blood count, hemoglobin, serum folate, vitamin b12, iron, and ferritin levels. their study showed statistical significance between the observed groups, with overall deficiencies amounting to 56.2% in patients with ras versus 7% in healthy age-matched individuals (p < 0.0001). when looking at anemia alone, 34.4% of diseased subjects had anemia, compared with 6.9% of healthy individuals. they implied that ulceration could anticipate the onset of anemia, which is why routine laboratory blood tests should be performed in patients with ras to promptly diagnose hematinic deficiency and prevent more severe clinical manifestations caused by iron, folic acid, or vitamin b12 deficiency. their study established a firm connection between positive family history and the onset and severity of symptoms. no significant differences were found in the body mass index in all observed groups. the second part of their research involved administering adequate replacement therapy for a month to patients with ulcers who had some hematinic deficiencies, after which they were re-evaluated in a 3-month follow-up period. their results strictly classified two groups of people, one with total remission of ulceration in the oral cavity and another without remission. the variable by which they were divided was family history of ras. people with negative family history completely recovered after replacement therapy, but in patients with positive family history, ulceration was present despite therapy. this fact emphasizes the importance of detailed case history examination and leads us to adequate therapeutic options for different patients with the same disease. sun et al. (14) piloted a 6-year study with 273 patients with ras and the same number of controls. this was the longest study with the highest number of participants regarding this issue. they showed statistically significant differences between the observed groups, with lower mean hemoglobin in patients with ras (p < 0.001) and lower iron levels for women in the diseased group (p < 0.001). male subjects also had lower iron levels, but this was not statistically significant. in addition, there was no seemedj 2022, vol 6, no 1 iron deficiency and recurrent aphthous stomatitis 110 southeastern european medical journal, 2022; 6(1) distinction in the levels of vitamin b12 and serum folate between groups. however, when adjusted in hemoglobin, iron, vitamin b12, or folic acid deficiency groups by the world health organization criteria, patients with recurrent stomatitis had a significantly higher frequency of observed hematinic deficiencies (20.9%, 20.1%, 4.8%, 2.6%, respectively) than healthy control subjects (p < 0.05). nevertheless, approximately 60% of people who suffer from recurrent aphthous stomatitis had blood test results within normal limits, which can be explained by genetic susceptibility and other possible causes, such as immunological, hormonal, and emotional factors. slebioda et al. (11) analyzed blood samples of 71 subjects with ras and 70 individuals without the disease. they measured hemoglobin, hematocrit, mean corpuscular volume, red blood cell count, iron, and vitamin b12 levels. when comparing those two groups, hematinic deficiencies were more common in patients with aphthae. the mean serum iron levels were lower in the ras group than in the control group (88.6 lg/dl vs. 105.88 lg/dl). however, there was no observed correlation between hematinic deficiencies and clinical presentation and the severity of aphthae in the tested sample. therefore, these results may easily describe a confounding factor, and not the actual cause of the disease. babaee et al. (28) conducted a research about oxidative stress in 28 patients with ras and the same number of healthy individuals. they compared markers of oxidative stress in the saliva and routine hematological tests in both groups. salivary malondialdehyde is the main product of lipid peroxidation and it indicates oxidative stress. the oral cavity is the first entry point for outside pathogens, which is why saliva contains variant antioxidants to help fight infection. their overall activity coincides with the total antioxidant capacity (tac). when comparing the group with ras and the healthy group, salivary malondialdehyde level was considerably higher (p < 0.001) and tac level was significantly lower (p < 0.042). the imbalance between oxidants and antioxidants may cause many diseases of the oral mucosa. several studies also acknowledge that tissue damage in patients with recurrent stomatitis is caused by disruption of the oxidant/antioxidant balance (29). according to hematological parameters, only the ferritin levels were significantly higher in ras patients (p < 0.008). ferritin is an indicator of the body’s iron reserves and can expedite oxidative stress. production of ferritin can initiate lipid peroxidation and the formation of reactive oxygen species (30). therefore, high serum ferritin in ras can be used as an inflammatory measure, comparable to creactive protein or erythrocyte sedimentation rate. another hypothesis is that ferritin may be a protective attribute by making chelation with free iron during prolonged oxidative stress (28). conclusion numerous controlled studies analyzing the hematological status and iron levels of patients with recurrent aphthous stomatitis have been conducted. however, the methods, number of participants, including criteria, referent numbers, gender distribution, and other factors are not standardized. therefore, the results remain inconclusive, with some authors reporting a strong connection between iron levels and the development of ras, while others disagree. nevertheless, routine hematological screening in patients with ras may help reveal hidden nutritional deficiencies and prevent related systemic manifestations. additional studies are required to improve comprehensive understanding and clarify the impact of serum iron levels on recurrent aphthous stomatitis. acknowledgement. none disclosure funding. none. competing interests. none to declare. seemedj 2022, vol 6, no 1 iron deficiency and recurrent aphthous stomatitis 111 southeastern european medical journal, 2022; 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27:229-32. doi: 10.1016/j.amjoto.2005.09.022. 24. piskin s, sayan c, durukan n, senol m. serum iron, ferritin, folic acid, and vitamin b12 levels in recurrent aphthous stomatitis. j eur acad dermatol venereol 2002; 16:66-7. doi: 10.1046/j.1468-3083.2002.00369.x. 25. al-amad sh, hasan h. vitamin d and hematinic deficiencies in patients with recurrent aphthous stomatitis. clin oral investig 2020; 24:2427-32. doi: 10.1007/s00784-019-03102-9. 26. lopez‐jornet p, camacho‐alonso f, martos n. hematological study of patients with aphthous stomatitis. int j dermatol 2014; 53:15963. doi: 10.1111/j.1365-4632.2012.05751.x. 27. compilato do, carroccio an, calvino fr, di fede g, campisi g. haematological deficiencies in patients with recurrent aphthosis. j eur acad dermatol venerol 2010; 24:667-73. doi: 10.1111/j.1468-3083.2009.03482.x. 28. babaee n, hosseinkazemi h, pouramir m, baboli ok, salehi m, khadir f, bijani a, mehryari m. salivary oxidant/antioxidant status and hematological parameters in patients with recurrent aphthous stomatitis. caspian j intern med 2016; 7:13-8. 29. bilgili sg, ozkol h, takci z, ozkol hu, karadag as, aslan m. assessment of the serum paraoxonase activity and oxidant/antioxidant status in patients with recurrent aphthous stomatitis. int j dermatol 2013; 52: 1259-64. doi: 10.1111/ijd.12084. 30. alsultan ai, seif ma, amin tt, naboli m, alsuliman am. relationship between oxidative stress, ferritin and insulin resistance in sickle cell disease. eur rev med pharmacol sci 2010; 14:527-38. author contribution. acquisition of data: am, nm, zb, kv, dv, ipm administrative, technical or logistic support: am, nm, zb, kv, dv, ipm analysis and interpretation of data: am, nm, zb, kv, dv, ipm conception and design: am, nm, zb, kv, dv, ipm critical revision of the article for important intellectual content: am, nm, zb, kv, dv, ipm drafting of the article: am, nm, zb, kv, dv, ipm final approval of the article: am, nm, zb, kv, dv, ipm provision of study materials or patients: am, nm, zb, kv, dv, ipm statistical expertise: am, nm, zb, kv, dv, ipm seemedj 2022, vol 6, no 1 deep ecology 129 southeastern european medical journal, 2022; 6(1) review article deep ecology: contemporary bioethical trends 1 sandra mijač 1, goran slivšek 2*, anica džajić 3 1 department of microbiology, molecular diagnostics unit, synlab croatia, zagreb, croatia 2 department of intensive medicine, anaesthesiology, intensive medicine and pain management clinic, clinical hospital centre rijeka, croatia 3 department of translational medicine, children’s hospital srebrnjak, zagreb, croatia *corresponding author: goran slivšek, goran.slivsek@xnet.hr received: oct 15, 2022; revised version accepted: feb 8, 2022; published: apr 27, 2022 keywords: bioethics, ecological and environmental concepts, sustainable development, one health, public health abstract deep ecology emphasizes the importance of the ecological problems as a practical issue, and its importance is in changing the human understanding of everything, including even man’s understanding of who he is. the aim of this paper was to present deep ecology, what it represents and how it has become a significant ecological movement of the 20th century and to indicate the connection between bioethics as new environmental ethics and deep ecology, as well as other environmental movements which, in the contextualization of bioethics, emphasize changing the outlook on life, giving a better knowledge of it, and allowing questioning of social actions and looking at events from different aspects. the idea is to emphasize that man is not only an active, but also a responsible being which is capable of making a paradigm shift in responsibility, and therefore, taking responsibility for all life on earth. content analysis and comparative method were introduced and applied for the requirements of making this review. based on the obtained results, the review points to the need to create new ethics which could introduce a general value system for all living and non-living things a paradigm shift involving man as part of nature and not opposed to it, and to successfully address these complex issues. it will take a profound shift in human consciousness to fully comprehend that it is not only plants and animals that need a safe habitat because they can live without humans, but humans cannot live without them. (mijač s, slivšek g, džajić a. deep ecology: contemporary bioethical trends. seemedj 2022; 6(1); 129139) seemedj 2022, vol 6, no 1 deep ecology 130 southeastern european medical journal, 2022; 6(1) introduction from the beginning of man’s life on earth, every invention and discovery he had made to ease life was about subduing nature for his benefit (1). the reason why the problem began to appear, back in ancient times, is the importance of the presentation of the course of human thought and how changing this thought has led to the consciousness that in its expression subjugated the entire world around itself (2). deep ecology emphasizes the importance of ecological problems as a practical issue, and its importance is in changing the human understanding of everything, including man’s awareness of himself (3). the result produced would be that deep ecology, pointing to the value of all living things, also wants to point to the responsibility that people have in their environment. the new ethics must also have the dimension of sustainability, which can be accomplished in the frame of bioethics, as an interdisciplinary area of science. it is necessary to change awareness so that people can re-establish a relationship with nature without perceiving nature as a resource from which man will have a (short-term) benefit (4). in that sense, international nature and environment protection laws are deficient in practice, and citizens also need to contribute to ecological awareness. by unifying human approaches in the relationship to nature, this review aims to show that this relationship has become threatened. the aim was to determine whether deep ecology finds its justification in the change of awareness regarding human relationship to people and nature and to show how and to what extent environmental and nature protection which exceeds ecology in its complexity is carried out. deep ecology scientists have the most significant responsibility when it comes to preservation and strengthening of the ethical principles in their research and institutions, to act beneficially upon this crossroad of fate from where one can either crash into eternal doom or finally get into the haven of peace (5). increased interest in the problem of the environment (i.e., the ecological problem) began to appear during the 1970s, and considering the need for new ethics, some scientists and ecologists came up with the idea of said ethic. that considered, rand aldo leopold, a forester, philosopher, writer, teacher, and one of the greatest american biologists called such ethics the ethics of the earth, which would, by expanding the boundaries of the community, contain everything from earth to animals (1). he explained the base of his ethics, which was to protect wholeness and stability, and only then can the righteousness of the matter itself be discussed. arne næss expanded the thought behind such ecological movement with the diversity between surface and deep elements, where the surface elements mark our avoidance to contaminate the environment exclusively for our own benefit. in contrast, the deep elements represent the protection of the whole biosphere, regardless of the benefits a human being could have (6). this division in the surface and deep elements, that is, shallow and deep ecology, points to the meaningful division within contemporary ecological thought (7). according to that, shallow ecology represents the anthropocentric thought in which a human being is above nature, and nature has only instrumentalist value, while deep ecology goes for the highest ecological norm: preservation of the vital needs of everything living (8). the maker of the term deep ecology, arne dekke eide næss, who was born in 1912 and died in 2009 in oslo (1). he was one of the most famous norwegian philosophers, who taught at the university of oslo between 1937 and 1970, where he also graduated and completed a master’s degree. he taught semantics and gathered a group of young philosophers and sociologists who were applying empirical methods to affirm the meaning of philosophical terms. he also taught the philosophy of science and the philosophy of spinoza and gandhi (9), who also had a significant impact on him. as a hiker and a tour guide of the first expedition to the tirich mir mountaintop in the islamic republic of pakistan, his motivation for nature and environmental protection was no wonder. seemedj 2022, vol 6, no 1 deep ecology 131 southeastern european medical journal, 2022; 6(1) although it is not about the motivation founded on the reformist current of the ecological movement, which only wants to prevent contamination, naess should be given a closer look as a supporter of the revolutionary current, who supports the original current, but who also builds his philosophy seeking for new metaphysics, cognitive theory, and ethics which would solve the relationship between a human being and nature. he called this (eco)philosophy, which is contained in the term deep ecology and synonymous with the terms fundamental ecology, a new philosophy of nature, ecosophy, or ecophilosophy t. in that regard, ecosophy t is built starting with oneself, the change within oneself – to act upon welfare as a whole (1). the core of næss’s philosophy is about connecting everything into a whole, that is, the idea that nothing works independent of the whole, meaning that the relationships between people, plants, and animals depend on one another. according to that, two fundamental principles of that philosophy stand out, as well as those of the ecological movement: self-fulfilment and biospheric equality (5). contrary to health and welfare of the population, more precisely the population which lives and acts in the developed industrial countries as a central theme of the contemporary society fighting against the contamination of the environment, næss turns to the inner knowledge of norms, values and ethics, meaning that ecological science will bleed into interdisciplinary practical life wisdom (3). naess called that transition deep ecology (9). furthermore, næss and the american philosopher george sessions (who also referred to the new ecological ethic which næss discovered in 1972 and referred to as deep ecology) shaped and exposed the principles which would work for the deep ecology platform, in eight chapters in an article from 1984. some of those principles are: 1. the welfare and the success of human and non-human life on earth have their own values (synonyms: intrinsic value, inherent value). those values do not dependent on the usefulness of the non-human world for humans. 2. the richness and diversity of life forms contributes to the realization of these values. people have no right to jeopardize that richness and diversity unless the goal is to satisfy life needs (6). the authors state that, although those principles relate to life when we talk about the term biosphere, they are also meant to include the unliving, like rivers, environment, and finally the ecosystem. naess replaces the term biosphere with the term ecosphere, and that way he does not limit himself to the form of life in the immediate or global surroundings (9). in addition, he replaces the term environment with the term co-world to mark the place of a human in the most truthful way possible. deep ecology increases the meaning of the principle of letting the being be (10) while trying to bring ecological consciousness to a higher level and achieve a healthier ecological life. among other things, deep ecology is founded on darwinist thought, which tries to move the human away from the centre of life and into a natural circuit of existing (9). because of that, the darwinist element presented in the deep ecology builds a complex and contradictory relationship. deep ecology postulates that exiting from evolutional and acceptable circumstances, which darwinism sets as an imperative in the way of life, damages the human civilization and nature (1). it exposes the human being and breaks the illusion that humans are wise enough to rationally manage their physical and social environment, not taking into account the evolutionary processes (9). another relevant characteristic of deep ecology is its attitude towards wilderness, the only realworld left, around which, because of its ecocentric orientation, exists a cult of wilderness (11). according to that, it advocates ecoregionalism and condemns urbanization and hypermobility. it is clear that deep ecology nearly revises that pantheistic belief and divinifies nature, but what needs to be underlined is that it does not replace religion, cults, or a mystical worldview, even though it has mystical aspects. the possibilities and the controversy of deep ecology are manifested even in its basic statement about the concept of intrinsic values, which states that every part of nature is valuable in itself, and not because of seemedj 2022, vol 6, no 1 deep ecology 132 southeastern european medical journal, 2022; 6(1) higher goals (human, for instance). in that regard, humans are a part of nature and not its highest achievement (9). however, nature is formed hierarchically, with humans on top, which subjects this concept to criticism and doubt (11). by replacing the term biospheric egalitarianism – in principle – with the term biospheric equality, næss equalizes all the organisms in the biospheric community, and their equality is a consequence of a relational interconnection, which gives them an intrinsic value. the fact that humans are at the top of the pyramid does not mean that they are not responsible for it. understanding that a human being must satisfy its needs to survive, næss does not deny those needs, but only for existential purposes, and when human secondary needs and vital needs of another species come into conflict, a human being should sometimes abandon egoism before the needs of other living beings (12). the authors of the book deep ecology, bill devall and george sessions, think that all organisms and entities in the ecosphere, as parts of an interconnected whole, are equal by intrinsic value. a question arises how all these living, but diverse beings are equal by their intrinsic value. furthermore, one criticism may be that even if there is an intrinsic value relating to the whole, the book does not say anything about the values of individuals. no individual is a necessity for the survival of the ecosystem as a whole (6). it is concluded that the ethics of the deep ecology does not answer the questions concerning the value of life of individual living beings. the reason may be that the wrong questions are being asked: ecological ethics might be more acceptable when applied to the level of species and ecosystem. in trying to establish that value based on the ecological ethics, a certain holistic feeling arises, a feeling that a species or the ecosystem is not just a total of individuals, but an entity in itself (3). authors like lawrence e. johnson, frey mathews, and james ephraim lovelock include species and ecosystems as holistic entities or selves with their own form of realization (6). if the species and the ecosystem can be considered a type of an individual with its own interest, the ethics of deep ecology must face the problems of determining the moral value of the species or the ecosystem again, regardless of the value which it has because of its importance for sustaining life (9). the fact that the biosphere can react to events in ways that look like a selfsustainable system does not show that the biosphere wants to contain itself consciously (1). this fact underlines that the ethics of deep ecology must reject its moral base because the argument stemming from the intrinsic value of plants, species, and the ecosystem is problematic (6). this, of course, does not mean that the argument for protecting intact nature is weak, but the argument based on the difference between the feeling and non-feeling creatures is firmer than the division between the living and non-living (5). the arguments should show that the value of preservation of the last significant areas of untouched nature significantly overcomes economic values (6). a human must acknowledge that value as an ethical category for that to happen, and therefore, confirm its responsibility (13). if a human’s realization of interests for his benefit is acknowledged as an intrinsic value, then it must also be acknowledged for other living beings who are ensuring their well-being (11). also, the concept of the “right of nature” is doubtful because it enters into a new manipulation. the right to preserve natural resources is contradictory to the concept of preservation of intrinsic values (13). the task of intrinsic values is building the marvel towards the wholeness of existence which is independent of humans (11). it stems from the fact that due to the prevalence of big cities and mechanicalized environment, such marvel cannot be seen or felt towards the non-human, which is what the deep ecology wants to revive. one of the objections to deep ecology is humanist voluntarism, which postulates that humans can change things by their own will. nevertheless, ecological destruction occurred because of actions of generations, and that is also why one generation cannot change it. the stumbling stone of deep ecology is that if it cannot change people’s awareness, it cannot lead to radical change (10). modern ecology states that nature existed before the first seemedj 2022, vol 6, no 1 deep ecology 133 southeastern european medical journal, 2022; 6(1) humans and that it will continue to exist, which is different from the understanding of tribal societies, and this is something that can be the encouragement for treating nature with more respect. tribal life, which deep ecologists advocate, is unacceptable for most people. in that regard, bioregionalism is unenforceable in the global world (11). talking about a relationship of a human being towards nature that is filled with awe, a german physician, theologian and philosopher ludwig philipp albert schweitzer is the most noted expert in defending ethics by expanding on sensitive beings (9). using the phrase “awe before life”, he builds the ethics of awe, which is based on having equal awe before every life, as well as one’s own life (11). he shaped the first and extensive attitude of philosophical biocentrism (7), but his ethics finds itself before the question: what is it like in the cases in which human life can be preserved only then when another human life has to be destroyed instead (14)? deep ecology sets a unique view of the relationship towards evolutionism. generally, the attitude of the deep ecologists is that modern life in industrial societies is not evolutionarily adjusted (11). tomislav markus understands that people did not kill nature, but they abandoned the environment of evolutionary adaptation. as the author points out, deep ecology is closer to science and philosophy, and it is not a moral lesson for wealthy individuals (10). markus points out that knowledge in biology and ecology is essential for understanding the relationship between humans and nature. so is the awareness of the pressure modern industrial societies put on the environment, which means that evolutionary adjustment to the environment is impossible. therefore, the author sets an imperative in creating a new view of nature, human nature, and human inadaptability to evolution (11). since the base of the humanist disciplines lies in dualism, a human as a being is separated from nature with its history about the self-creative process, which is founded on biophobia and ecophobia. the solution is found in the human need to escape into the circumstances of an organic existence (9), representing the escape from environmental destruction. according to that, deep ecology is the escape from consumerism, hyperurbanism, hyperpopulation, and all other significantly destructive orders of the modern industrial society (15). the solution might be seen in accepting naturalness as a characteristic of human nature, which could decrease environmental destruction. to stop environmental destruction, in favour of life preservation, deep ecology emphasizes the change of the paradigm (1). that would mean that the paradigm, which positions the human being in a superior position looking at nature exclusively as a resource, should change by accepting the evolutionary insights about people’s lives. it is trying to rise above consumerism as one of the characteristics of technical civilization. markus thinks that there are too many people living on this earth who are not one with nature and who, by that, challenge it by destruction (11). the solution is in the tribal communities, and the precondition is decreasing the population. it is the tribal communities who have the lowest rate of intervention in the environment, as opposed to industrial societies which replace life through the technical and, by doing so, they put pressure on the environment. according to næss, the quality of life of an individual and of an entire population cannot be considered if the size of that population is excessive. he agrees with decreasing the population in a non-violent way through voluntary birth control (12). also, he thinks that there should be a 100 million people less on earth. numerous deep ecologists believe that diseases, wars, and lack of food will more likely lead to decreasing the population than the rational, controlled way (10). for instance, when næss wrote about the solutions for depopulation, there were six billion people in the world, while today that number has exceeded seven billion and is still growing. as partially shown before, the two attitudes were determined according to ecoethics: shallow and deep ecology, which try to solve the problems regarding human violations against nature (16). various ecological ethics or ecoethics appeared because of the care for nature and the paradigm change, as is the case with deep ecology. deep ecology, by pointing to the value of all living and seemedj 2022, vol 6, no 1 deep ecology 134 southeastern european medical journal, 2022; 6(1) non-living beings, also wanted to indicate the responsibility of all towards the environment (17). that term, as well as others, lay the foundation of bioethical principles, and the relationship between bioethics and deep ecology (5). bioethics and deep ecology bioethics is a term that came into use in the 1970s, relating to ethical questions in the areas of biology, medicine and psychology in order to provide answers to the challenges of new knowledge. although the term bioethics, i.e., “bioethik”, was first used by paul max fritz jahr in an article from 1927, the credits for conceptualizing and preparing the term go to van rensselaer potter ii, who built the foundation for the development of bioethics in his work in the 1970s (15). since the meaning of life is broader than the human or medicinal aspect, bioethics questions the responsibility of human action towards humans themselves, but also towards all life on earth, or better said towards the biosphere (18). namely, potter thought that ethical values cannot be separated from biological facts, and he considered bioethics to be a bridge between science and humanity (19) which includes all living beings or, in other words, a biosphere essential for guaranteeing a future (20). numerous discoveries have brought new knowledge, which he believed could not in itself be completely bad or good, but that it represented power, and, therefore, once available, it would mostly be used for power (21). it is therefore essential to know how to use new knowledge, and that is possible only by possessing the wisdom on how to use new knowledge (22). on that end, he believed that bioethics as a science of survival would provide the wisdom on how to ensure sustainability (21). however, despite that, bioethics is often synonymous with clinical, medical or, the commonly called, biomedical ethics, which is wrong and inconsistent with potter’s original idea of a global bioethics which deals with man’s relationship with himself, but also with the ecosystem (23). bioethics cannot be only clinical ethics because the concept simultaneously contains elements of environmental ethics — it is concerned with the survival of man, but not any survival the survival which considers the survival of the ecosystem that has its value, entirely independent of man (24). finally, according to potter, bioethics implies the inevitable interconnectedness of man and the rest of the living world (25), or in other words, an interconnected biosphere (20). deep ecology as a part of environmental ethics understands people as an indispensable part of nature or a link in the chain of life, it points to the interconnectedness and interdependence of all parts of the ecosphere, emphasizes the primordial value of all species regardless of human needs, and it focuses on wisdom and balance (26). deep ecology can be seen as a form of a radical environmental critique of the technological civilization which reacts to technolatry, anthropocentrism, instrumentalism and resourcism, consumerism, and linear progressivism which overtook society with the emergence of new knowledge (27). naess considered deep ecology to be an ecosophy developed under the influence of leopold, focused on wisdom, that is, the wisdom of the earth, which focuses on ecologically wise and healthy living (28). it is shown that ecological ethics, ecoethics, or environmental ethics gather different theories, some of which are mentioned here. for example, ecocentrism, biocentrism, pathocentrism, or their mixed forms such as ecocentrism and ecofeminism, as well as the ethics of deep ecology from which each of them stems, try to set a frame in order to discuss the moral relationship between humans and inhuman entities, by expanding the human moral obligation to animals, plants or certain areas of nature or life in general (29). despite the critics and the deficiencies to which deep ecology subjected, the framework for building a new theory is the concept of responsibility, more precisely the responsibility of acting, as in lighting the effects of knowledge (30). also, new ethics must have a dimension of sustainability, which bioethics as an interdisciplinary field of science can realize within the scope of its content, and its strength can be seen in generating a new sensibility and creating a new awareness which goes past particular seemedj 2022, vol 6, no 1 deep ecology 135 southeastern european medical journal, 2022; 6(1) dimensions and tries to preserve life to stabilize all the segments of society (29). in the works of leopold and potter, it is evident that bioethics and environmental ethics share a common source. the connection between bioethics and deep ecology as part of the environmental ethic is in their vision of an interconnected biosphere (20). people are a part of the natural world, and not just bystanders, and based on that, the responsibility towards the world around and towards each individual is evident (31). bioethics and environmental ethics also share wisdom as a common root (21), mostly because of new knowledge. it is precisely because of that high complementarity between bioethics and environmental ethics that, in 1988, potter proposed the introduction of the new term global bioethics (32). potter coined the term global bioethics in an attempt to protect the new science of survival from a growing transition into a predominantly clinical ethics, but also to further expand it with even more elements of environmental ethics, especially under the influence of leopold’s legacy (33). however, despite all that, bioethics and deep ecology have over time developed into two separate fields (20), which has led to the creation of a gap between bioethics and environmental ethics (34). namely, bioethics has mostly developed into clinical ethics, where the focus is on the individual health of a human patient, while environmental ethics has developed more with the focus on biosphere health and not on individual health, that is, on the health and sustainability of the overall ecosystem (35). public health ethics as a bridge back to potter’s bioethics public health ethics is a relatively new field, coming into its own somewhere at the beginning of the 21st century, and it is still in its developmental stage but in recent years it has become one of the fastest growing subdisciplines of ethics (34). it is deeply rooted in bioethics, clinical and research ethics, and also in environmental ethics (36). public health ethics is primarily focused on policies, programs and laws for the protection and promotion of public health, and the focus is not on the individuals but on the community (i.e., the population) when it comes to achieving the common good (34). since health is a state of complete physical, mental and social well-being and not merely the absence of diseases or infirmity (20), the complexity of public health, and thus of public health ethics, is evident. the fact that human health depends on the environment has been known since the beginning of time, and today it is increasingly clear that it also depends on animal health, because the convergence of humans, animals, and their products is more pronounced than ever before (37). the current coronavirus pandemic shows the importance of interconnectivity of the domains of people, animals, and the environment as a group of interconnected circles when it comes to public health, but also when it comes to the future of all living things (38). severe acute respiratory syndrome coronavirus 2 is most likely the product of ecological conditions created by humans, while the related pandemic is a product of the number, density, and connectivity of the human species and its interaction of the environment (39). it is obvious that the health of humans is connected to the health of animals and the environment, and, therefore, we can say that the health of each of those three domains is the product of interactions of triangles of their health which, in fact, forms public health (40). that kind of public health – the one health approach (41) – is in line with potter’s vision of an interconnected biosphere; hence it can be considered as a planetary vision of one health (42) or global one health, and, consequently, we can talk about the global public health ethics (figure 1) (43). seemedj 2022, vol 6, no 1 deep ecology 136 southeastern european medical journal, 2022; 6(1) figure 1 the expanded model of the global one health concept potter sought to include health, survival, and the environment in the new ethics, which will combine knowledge and deliberation in the human constant quest for wisdom, that is, the knowledge of how to use new knowledge for the survival and progress of humankind (44). those qualities are contained and encouraged by public health ethics, which on one hand overlaps with bioethics, and on the other hand with deep ecology as part of the environmental ethics, while in its origins contains features of global ethics (20). public health ethics shows that human health is strongly and inseparably linked to the health of the planet (the biosphere) and that the health of the community is essential for the health of individuals, which in turn has a strong impact on the health of the population (45). that is not surprising since public health deals with the health of the individual, but also with the health of the environment, in order to achieve the best possible health of the population (20). the case of the coronavirus pandemic underlines the need for a fundamental shift in the human conception of health, sustainability, and humanity, which is only possible by returning to potter’s bioethics, which evaluates and considers all living beings, or in other words, the biosphere (46). based on everything mentioned above, public health ethics can be used to bridge the gap between bioethics and deep ecology as part of the environmental ethics to restore the values of potter’s bioethics for a brighter future of all living things (34). conclusion the history of ecology starts with the neolithic revolution, although it seems that it was only after the revolution that we heard about ecological problems. it has been confirmed that, at the same time when the human seemedj 2022, vol 6, no 1 deep ecology 137 southeastern european medical journal, 2022; 6(1) anthropogenic activities started to change his organic and wild environment, to which he is genetically adjusted, began the alienation of the wilderness that he has gotten used to (13). of course, it was not just humans who conditioned the (negative) changes in nature; there were also volcanic eruptions, asteroid collisions, earthquakes, and floods – in other words, a multitude of natural disasters to which most of the living world is not adjusted and most of which happened long before human existence. with the development of civilization, the shaping of cultures, and usage of technology, human beings genuinely become active factors in affecting nature. from greek philosophy to cartesianism, nature was thought to be the starting point for questioning everything (47). experiencing nature as a devalued magnitude and the subject of knowledge conditions the forming of new things, more specifically new age humans. the new age products are modern science and technology, in which science is the beholder and technology is the executioner (48). the role of technology is to satisfy the needs of life as quickly and pleasingly as possible, and through that, the consumer society is created, which also affects the expansion of the ecological crisis. it is no wonder that the relationship of a human being and nature is altered because of the eternal nature of modern science and technology (49). numerous archaeological studies have shown that the ecological problems started with the neolithic domestication, which has increased in intensity in the last few centuries and led to an ecological crisis (50). although the ecological crisis does not affect everyone equally, it is a problem that significantly influences life and demands an urgent solution, regardless of those who think that the ecological crisis is either a reflection of capitalism or industrialization, contrary to those who believe that technology could solve the problems of humanity (51). ecology contains many areas affected by biosphere processes, which should be contained to access the solution to its problems. this should be done with the help of sustainable development, which presents the principles of sustainability of the system, a way of development that does not degrade or violate nature (50). however, to achieve progress, it is people’s attitude towards nature that must change, not their attitude towards themselves, which is how næss formulated it in his philosophy, known under many other terms, but mentioned here most often under the term “deep ecology” (52). the concept that emphasises the value of every life — in (new) bioethics, ethics of life, which due to its interdisciplinary area of impact can be applied in reality, is enriched through that responsibility (53). in recent years, it has come to light that public health ethics can be used to bridge the gap between bioethics and deep ecology as part of the environmental ethics, thus enabling the return to potter’s bioethics which has built-in values of deep ecology (54). although much has been done in recent years, deep ecology is to a great extent still in its very beginnings. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. bhaskar r, høyer kg, næss p, editors. ecophilosophy in a world of crisis: critical realism and the nordic contributions. london: routledge; 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3(1): 83-93. doi: https://doi.org/10.3126/sjah.v3i1.35377 52. valera l. el futuro de la ecología: la sabiduría como centro especulativo de la ética ambiental [the future of ecology: wisdom as the speculative centre of environmental ethics]. cuad bioet. 2016; 27(91): 329-338. spanish. 53. dwyer j. how to connect bioethics and environmental ethics: health, sustainability, and justice. bioethics. 2009; 23(9): 497-502. doi: 10.1111/j.1467-8519.2009.01759.x 54. goldberg tl, patz ja. the need for a global health ethic. lancet. 2015; 386(10007): 37-39. doi: 10.1016/s0140-6736(15)60757-7 author contribution. acquisition of data:sm, gs, ad administrative, technical or logistic support: sm, gs, ad analysis and interpretation of data: sm, gs, ad conception and design: sm, gs, ad critical revision of the article for important intellectual content: sm, gs, ad drafting of the article: sm, gs, ad final approval of the article: sm, gs, ad seemedj 2021, vol 5, no. 1 upper extremity rehabilitation and neurologic impairment 96 southeastern european medical journal, 2021; 5(1) original article effects of robot-assisted upper extremity rehabilitation on change in functioning and disability in patients with neurologic impairment: a pilot study 1 valentina blažinčić *1, 2, ivica ščurić 1, 3, ivana klepo 1, ivan dubroja 1, duško cerovec 1, 2 1 special hospital for medical rehabilitation krapinske toplice, croatia 2 faculty of medicine, josip juraj strossmayer university of osijek, croatia 3 school of medicine, university of zagreb, croatia *corresponding author: valentina blažinčić, valentina.blazincic@sbkt.hr received: oct 18, 2021; revised version accepted: mar 15 2021; published: apr 28, 2021 keywords: robotics, upper extremity, international clasification functioning, disability and health, traumatic brain injuries, stroke abstract introduction: the aim is to evaluate the effect of robot-assisted training on the most important aspects of functioning and disability in patients with upper extremity neurologic impairment. materials and methods: a prospective six-week pilot study included robot-assisted training of the upper extremity and conventional neurorehabilitation in 12 participants after a stroke or traumatic brain injury. outcome measurements were range of motion (rom), the international classification of functioning, disability and health (icf) core set for hand and the visual analog scale (vas) for pain sensation. a wilcoxon test was used for the analysis of preand post-test differences and spearman’s correlation was used for connecting the data collected. results: a statistically significant difference was found for rom (shoulder abduction/adduction, shoulder flexion/extension, shoulder internal/external rotation and forearm pronation/supination) and a number of icf categories (body function: b280, b710, b715, b730, b760; activities and participation: d230, d430, d440, d445, d5). a significant positive correlation of medium intensity (r=0.589) was found between the duration of movement coordination training and the icf category b760. we did not find a statistically significant difference in pain sensation (vas) with regard to the direct use of the device. for all analyses, p<0.05 and ci was 95%. conclusion: robot-assisted training and conventional neurorehabilitation improved motor and functional recovery. there was a correlation between training a specific goal on the device and one of the icf body function categories. (blažinčić v, ščurić i, klepo i, dubroja i, cerovec d. effects of robot-assisted upper extremity rehabilitation on change in functioning and disability in patients with neurologic impairment: a pilot study. seemedj 2021; 5(1); 96-108) seemedj 2021, vol 5, no. 1 upper extremity rehabilitation and neurologic impairment 97 southeastern european medical journal, 2021; 5(1) introduction advances in medicine are impossible without the combined effort of advances in different technologies. today, the use of novel technology in medicine is expected. since 2011, an upper extremity exoskeleton has been commercially accessible for rehabilitation (1). but how can we recognise and subsequently combine the clinical needs of patients with the corresponding devices (2)? robot-assisted devices for the upper extremity have been created for training and assisting or can combine these two functions (3). according to the world health organization, stroke, traumatic brain injury (tbi) and spinal cord injury (sci) are defined as chronic diseases (4). while various neurological conditions affect different populations and have different pathophysiology, they all damage neural networks and motor system networks in particular. as such, clinical impairment and functional problems of persons with different neurological conditions can often overlap (5) and analysing them together may be of interest (6). around 70% of patients with stroke (7) and 30% of patients with tbi exhibit upper extremity paresis at rehabilitation admission (8,9). for restoration of upper extremity function in patients with a neurologic deficit, the main therapy strategies target the impaired motor cortex (activation of ipsilateral or inhibition of contralateral) or affect afferent sensory pathways (10). it is well-recognised that an intense training program during rehabilitation for patients in the subacute period (i.e. in the first 6 months) will significantly improve functional outcomes for the upper extremity (10.11). metaanalyses have demonstrated significant effects on motor control and muscle strength using shoulder/elbow robotics, as well as on motor control using elbow/wrist robotics (12). for persons with neurological deficits in a chronic phase, robot-assisted rehabilitation is more effective than other types of therapies for recovery of upper extremity motor function (13). together, these results indicate that the use of an exoskeleton device with various possibilities (virtual reality, augmented reality and gamification) lead to general improvement of motoric function in neurorehabilitation (14). but can it lead to an improvement in overall functional status? in the present study, we decided to use the international classification of functioning and disability (icf) as a measurement tool in light of the notion that limitations of function and disability are not only related to aetiology, but can also be considered as general manifestations in overall health conditions (15). the icf also has other advantages in its use of a common language (i.e. it is internationally comparable) (16) and has the essential and most relevant categories for personal functioning when using the icf core set information (17). the first aim of this investigation is to assess the efficacy of robotassisted training on the motoric function of the upper extremity. secondly, this study aims to examine change in functioning and disability among persons with upper extremity impairment caused by different neurologic aetiology at a clinical level during inpatient rehabilitation. the first expected effect of robotassisted training is an increase in the range of motion (rom) of joints in the trained upper extremity and changes in activity and participation among the participants. the second expected effect is a connection between the time spent in training specific goals on the robot-assisted device and selected body domains of the icf core set. pain sensation in the trained extremity will also be evaluated. materials and methods this prospective pilot study was conducted at the special hospital for medical rehabilitation krapinske toplice in croatia. prior to the commencement of the study, informed consent was obtained from all participants and the study was conducted according to the helsinki declaration. consent for publication of the study results was obtained from the ethical committee of the special hospital for medical rehabilitation krapinske toplice.twelve participants (3 women and 9 men) aged between 20 and 61 years who underwent acute neurorehabilitation following a traumatic brain injury or stroke met the inclusion seemedj 2021, vol 5, no. 1 upper extremity rehabilitation and neurologic impairment 98 southeastern european medical journal, 2021; 5(1) criteria. inclusion criteria were: aged 18 years and older, paresis of the upper extremity following a traumatic brain injury or stroke, ability to sit (i.e., exhibits trunk control in a sitting position) and a rancho level of function of 5 or more. exclusion criteria were: non-acute rehabilitation phase, paresis of the upper extremity of other aetiology, plegia of the upper extremity, inability to sit (i.e., exhibits inadequate trunk control in sitting position), fixed contractures of shoulder, elbow or wrist joints, a rancho level of function less than 5, apraxia, hemineglect and receptive aphasia. the study was designed as a six-week program. participants received robot-assisted training during their regular occupational therapy sessions for 30 minutes, 5 days a week. all participants were assessed at the beginning and at the end of the study. our primary outcome measurements were rom (for shoulder and elbow joints) and categories of the icf core set hand conditions brief (for the trained upper extremity). the evaluation of pain sensation in the trained upper extremity was a secondary outcome measurement. for the assessment of rom, kinematic parameters of the shoulder and elbow joints (i.e. minimal and maximal shoulder abduction/adduction, minimal and maximal shoulder flexion/extension, minimal and maximal inner/outer shoulder rotation, minimal and maximal elbow flexion/extension and minimal and maximal forearm pronation/supination) recorded during training on the robot-assisted device were collected and analysed. assessment of the selected icf core set was conducted by one investigator following additional education. the icf core set is made up of 23 categories (9 in body function, 3 in body structures, 8 in activities and participation and 3 in environmental factors). every category was assessed using the icf qualifiers on an ordinal scale ranging from 0 (no impairment) to 4 (complete impairment), with additional possible values of 8 (not specified) and 9 (not applicable). for most categories, assessment was carried out during clinical examination of the participants, while for some categories, information from a patient-report questionnaire was sufficient. some categories also have additional qualifiers: body structures 2 (for nature and location of change), activities and participation 2 (for capacity and performance) and environmental factors (an). the evaluation of pain sensation was also carried out by one investigator. to assess pain directly related to the use of the device, the visual analog scale (vas) was conducted immediately before and after the first and last training session on the robot-assisted device. prior to the beginning of the study, three occupational therapists were trained to use the robot-assisted device. an exoskeleton device, the armeo®spring hocoma, inc., composed of an upper and lower module for the upper arm and the forearm and a pressure sensitive handgrip for the hand was used (18). both modules were length-adjustable, which allowed adequate positioning of the exoskeleton device and the arm joints of the participants. in addition, the weight compensation feature of exoskeleton (18) was utilised and individually adjusted for all participants. using this exoskeleton device, our participants were enabled to train the shoulder and elbow joints, flexo-extension of the wrist and manual grip (19). the repetition of movements of the paretic upper extremity during virtual gaming was conducted within a three-dimensional workspace (18). the goals of robot-assisted therapy were to increase rom (1d and 2d/3d), to improve movement coordination and to improve grasp function and cognitive training. all participants also underwent conventional neurorehabilitation (i.e. they received therapies individually indicated according to their overall clinical status, which included a multidisciplinary approach if needed).. statistical analysis statistical evaluation was performed using spss, version 26.0 (spss inc., chicago il, sad). in the first analysis, a wilcoxon signed-rank test was used to evaluate preand post-test differences for all data collected (kinematic parameters, icf categories and vas). statistical significance was set at p<0.05 and ci was 95%. all quoted p-values are two-tailed. in the second seemedj 2021, vol 5, no. 1 upper extremity rehabilitation and neurologic impairment 99 southeastern european medical journal, 2021; 5(1) analysis, we used the spearman’s correlation coefficient to test relationships in the data (duration of training a specific therapy goal on the robot-assisted device and selected icf categories). a correlation greater than r>0.80 was considered as strongly positive, 0.5<r>0.8 was considered as medium to strongly positive and 0<r<0.5 was considered as weakly positive. a correlation coefficient greater than 0.5, r> 0.5 was considered significant. results all 12 participants (3 women and 9 men) successfully completed the pilot study. seven participants (58.3%) exhibited neurologic impairment following tbi and 5 participants (41.7%) following stroke. the average age of participants was 39.42 years (standard deviation (sd) 16.94) and ranged between 20 and 61 years. the right arm was treated in half of the participants and all participants exhibited righthand dominance. the average number of therapy sessions with the robot-assisted device was 25.50 (sd 5.98), where the number of sessions ranged between 14 and 33. the average therapy time using the robot-assisted device per treatment was 13.75 minutes (sd 2.67), ranging from 8.74 to 16.84 minutes. from the total number of therapies using the exoskeleton device, the average time spent performing each exercise was: 5.05 minutes (sd 1.21) for increasing rom 1d, with a minimal value of 3.16 and a maximal value of 7.43; 6.48 minutes (sd 1.86) for increasing rom 2d/3d, with a minimal value of 4.26 and a maximal value 10.86; 9.41 minutes (sd 2.86) for movement coordination, with a minimal value of 5.30 and a maximal value of 15.58; 2.88 minutes (sd 1.75) for grasp function, with a minimal value of 0.09 and a maximal value of 6.76; and 2.25 minutes (sd 1.09) for cognitive training (11 participants), with a minimal value of 0.09 and a maximal value of 3.50. primary outcomes kinematics parameters a wilcoxon test showed statistically significant differences between the initial and final measurements for the following kinematics parameters observed: minimal shoulder abduction/adduction, shoulder abduction/adduction range of motion, minimal shoulder flexion/extension, shoulder flexion/extension range of motion, minimal shoulder internal/external rotation, maximal shoulder internal/external rotation, shoulder internal/external rotation range of motion, maximal elbow flexion/extension, minimal forearm pronation/supination and forearm pronation/supination range of motion. ci was 95% (table 1). icf core set -hand conditions brief a wilcoxon test demonstrated a statistically significant difference between the initial and final assessment using the icf core set for the following categories in body function: b280 (sensation of pain), b710 (mobility of joint functions), b715 (stability of joint functions), b730 (muscle power function), b760 (control of voluntary movement function). similarly, statistically significant differences were found for the following categories in activities and participation: d230 (carrying out daily routine), d430 (lifting and carrying objects), d440 (fine hand use), d445 (hand and arm use) and for d5 (self-care). ci was 95% (table 2). seemedj 2021, vol 5, no. 1 upper extremity rehabilitation and neurologic impairment 100 southeastern european medical journal, 2021; 5(1) table 1. data of the kinematics parameters (ranges of motion in degrees) for the upper extremity joints presented as test statisticsa variable name value before (degrees), x̅±sd value after (degrees), x̅±sd p-value shoulder abd./ ad. min. -71.14±18.82 -88.12±3.24 0.009 shoulder abd./ad. max. 16.09±13.50 22.61±14.29 0.117 shoulder abd./ad. range 86.41±26.44 110.73±16.09 0.028 shoulder flex./ext. min. 52.90±3.87 50.22±2.05 0.019 shoulder flex./ext. max. 107.34±17.98 112.34±21.59 0.060 shoulder flex./ext. range 54.44±18.76 62.12±21.90 0.023 shoulder int./ext. rotation min. 32.98±18.88 18.16±17.79 0.019 shoulder int./ext. rotation max. 75.36±20.84 106.14±20.14 0.012 shoulder int./ext. rotation range 42.38±18.43 87.98±24.45 0.003 elbow flex./ext. min. 22.72±15.32 18.62±15.81 0.241 elbow flex./ext. max. 94.77±11.86 102.22±6.17 0.025 elbow flex./ext. range 72.05±21.98 83.60±19.12 0.062 forearm sup./pron. min. -23.52±40.66 -48.74±27.39 0.004 forearm sup./pron. max. 55.45±13.19 58.49±9.29 0.285 forearm sup./pron. range 78.97±37.7 107.24±23.53 0.012 a. wilcoxon signed-rank test p <0.05 was considered significant. abd./ab.= abduction/adduction; flex./ex.t=flexion/extension; int.=internal, ext.=external, min.=minimal; max.=maximal seemedj 2021, vol 5, no. 1 upper extremity rehabilitation and neurologic impairment 101 southeastern european medical journal, 2021; 5(1) table 2: data of the assessed icf core set presented as test statisticsa, p<0.05 was considered significant. variable name value before x̅±sd value after x̅±sd p-value b152 (emotional functions) 1.50±0.52 1.42±0.52 0.317 b265(touch function) 0.83±0.94 0.75±0.87 0.317 b270 (sensory functions of sensing surfaces and their texture or quality) 0.17±0.39 0.08±0.29 0.317 b280 (sensation of pain) 1.0±0.95 0.58±0.79 0.025 b710 (mobility of joint functions) 1.92±1.24 1.17±1.12 0.014 b715 (stability of joint functions) 1.25±1.48 0.75±1.22 0.034 b730 (muscle power functions) 2.42±0.80 1.75±0.75 0.005 b760 (control of voluntary movement functions) 2.25±0.97 1.50±0.67 0.014 b810 (protective functions of the skin) 0.08±0.29 0.00±0 0.317 d230 (carrying out daily routine) 2.67±1.07 1.92±0.90 0.003 d430 (lifting and carrying objects) 2.751±1.14 2.17±0.84 0.008 d440 (fine hand use) 2.58±0.90 1.92±1.00 0.011 d445 (hand and arm use) 2.33±0.65 1.50±0.52 0.002 d5 (self –care) 2.42±1.09 1.83±1.12 0.020 d7 p (interpersonal interactions and relationships) 1.58±0.90 1.67±0.89 0.317 d7 c (interpersonal interactions and relationships) 1.08±0.79 1.08±0.79 1.000 e1 (products and technology) 2.25±0.75 2.58±1.08 0.194 e3 (support and relationships) 2.75±1.14 2.58±1.96 0.785 e5 (services, system and policies) 1.83±1.12 1.00±1.35 0.655 a. wilcoxon signed-rank test seemedj 2021, vol 5, no. 1 upper extremity rehabilitation and neurologic impairment 102 southeastern european medical journal, 2021; 5(1) secondary outcomes data connectivity spearman’s correlation coefficient demonstrated a significant positive correlation of medium intensity, between the duration of the movement coordination therapy session using the robot-assisted device and the icf category b760, indicating a positive relationship between these variables r=0.589 (p-value of 0.044) (figure 1). no significant correlation between the duration of the therapy session for increasing rom (1d and 2d/3d) and the icf category b710 was found (table 3). pain assessment a wilcoxon test did not indicate any statistically significant difference in pain sensation for the trained upper extremity, as measured with vas. ci was 95% (table 4). figure 1: connection between therapy time for movement coordination and the icf domain b760) (difference in value of control of voluntary movement functions) as measured by spearman’s correlation coefficient *correlation is significant at the 0.05 level (two-tailed). table 3. connection between therapy time, rom 1d, rom 2/3d and the icf domain b710 (mobility of joint functions), as measured by spearman’s correlation coefficient. variable name therapy time rom 1d rom 2/3d b710 (difference) therapy time r 1.0 0.21 0.33 -0.21 rom 1d r 0.21 1.0 0.13 -0.35 rom 2/3d r 0.33 0.13 1.0 -0.5 b710 (difference) r -0.21 -0.35 -0.5 1.0 *correlation is significant at the 0.05 level (two-tailed). rom 1d=range of motion, one dimension; rom2/3d=range of motion, two and three dimension icf=international classification of functioning, disability and health. table 4. : test statisticsa for pain sensation data, p<0.05 was considered significant variable name value before value after p-value vas i 13.83±24.53 10.42±13.89 0.385 vas ii 9.17±13.95 15.33±18.36 0.327 a. wilcoxon signed-rank test vas i= sensation of pain immediately before therapy session using the device. vas ii= sensation of pain immediately after therapy session using the device. seemedj 2021, vol 5, no. 1 upper extremity rehabilitation and neurologic impairment 103 southeastern european medical journal, 2021; 5(1) discussion this pilot study confirmed the hypothesis that the use of a robot-assisted device in the rehabilitation of patients with paresis of the upper extremity leads to increased rom in joints of the trained upper extremity. other studies have also demonstrated motor improvement (rom, strength or motor control) following robot-assisted training in persons with neurologic impairment (20-23). the added advantage of the robot-assisted device as an assessment tool (24) was used for in this study for measuring rom in order to attain objective, quantitative data while avoiding the subjectivity of the investigator (25-27). in addition, another feature of the device – the ability to conduct a large number of repetitions – was used for improving rom. in a study by lo and colleagues, over 1,000 repetitions per session were achieved during a single hour of robot-assisted therapy (28). because motor recovery in neurologic impairment is considered to depend not only on cns damage, but also on the intensity (29) and duration of therapy, the ability to achieve a large number of repetitions in treatment is particularly important. in other words, more intense therapy is positively related to clinical improvements (30). the results of our study demonstrating a significant correlation between the duration of movement coordination therapy using the device and the icf body category that describes control of voluntary movement are consistent with this evidence. but what happens when participants exercise rom? the results of a neuroradiology study demonstrated that, when the sensation of movement is induced, the somatosensory, primary and supplementary motor areas of the cortex are activated and different proprioceptive inputs are associated with differently located activation patterns in these cortical areas (31). however, another important question is whether the patient will transfer this acquired motoric knowledge to everyday life? some investigators have developed and tested new strategies that aim to facilitate the transfer of new motor skills to everyday activities following robot-assisted training (32). in this study, we used the icf core set to understand and measure limitations in functioning and disability among our participants (33, 34) and to examine change following robot-assisted upper extremity training. the results demonstrated significant changes in various categories of body function and activity and participation. specifically, participants exhibited significant improvement in motoric function (as measured by the robotassisted device and the specific icf core set category) and had fewer limitations in functioning during inpatient rehabilitation (as measured by the icf core set categories). a study by goljar and colleagues demonstrated that the icf categories have the potential to reveal time-related changes in a patient’s functioning (35). the icf is also considered a useful framework for recognising the possibilities offered by devices in clinical or research settings (15). our findings are consistent with those of others who have investigated the effects of robot-assisted training on functional recovery. in a study by colomer and colleagues, significant improvement was found on both function and activity scales for the upper extremities, as measured by the motricity index, the fugl-meyer assessment scale, the motor assessment scale, the manual function test and the wolf motor function test (36). in another study using the fim as a measurement tool, daunoraviciene and colleagues found significant upper extremity improvement among participants who trained with a robotic device, as compared with a control group (37). the update of the cochrane review by mehrlotz and colleagues not only demonstrated improved muscle strength and function of the arm, but also improved scores for daily activities following the use of robot-assisted and electromechanical training in rehabilitation for persons after stroke (38). in regard to pain sensation, we did not find a significant difference related to the direct use of the exoskeleton device. in a study by busching and colleagues, semi-autonomous training was used for patients with severe paresis and no side effects were found with regard to training using the same type of the device (39). it is possible that our participants did not experience changes in pain sensation immediately after the use of the exoskeleton because the degree to which the seemedj 2021, vol 5, no. 1 upper extremity rehabilitation and neurologic impairment 104 southeastern european medical journal, 2021; 5(1) upper extremity was unweighted was individually adjusted for all participants with the intention of adequately supporting the paretic extremity and therefore facilitating the number of repetitions (40). however, when pain sensation is considered independently of the direct use of the exoskeleton device, the results of the icf core set demonstrated a significant change in the domain b280 (i.e., pain sensation decreased for participants during the six-week study period). findings from a double-blind randomised control study conducted by taveggia and colleagues also demonstrated a decrease in pain sensation over a six-week study period for both a robot-assisted group and a control group (41). in our study, the degree of spasticity was not considered and participants with upper extremity contractures (including those related to spasticity) were not included in the study in accordance with the exclusion criteria. the main limitation of this pilot study is the lack of a control group due to a small number of participants who met the inclusion criteria. as such, the question remains as to the degree of spontaneous recovery that occurred, which is difficult to distinguish from therapyinduced recovery without a control group for comparison. we also did not take into consideration new sensorimotor interactions between the exoskeleton device and the participants (42). muscle activity and muscle coordination is different in healthy persons (43, 44) when compared to persons with cns damage (pathological muscle synergies and altered joint coordination) and, because the exoskeleton device has its own mechanical characteristics and there is kinematic incompatibility during the human-exoskeleton interaction (45), currently the intention is to have as little interference as possible between the exoskeleton and the human body. in order to be as ergonomic as possible, new devices aim to be made on principles very similar to functional anatomy (46). conclusion robot-assisted training and conventional neurorehabilitation improved the motor and functional recovery of patients with upper extremity paresis of various aetiology. we consider the positive connection between the time spent training a specific goal using the robot-assisted device and one of the icf body function categories to be particularly important evidence. furthermore, improvement in motoric function achieved by affecting afferent sensory pathways might be responsible for improvement in the activity and participation category. in order to develop adequate recommendations for the use of robot-assisted devices in accordance with person-specific rehabilitation goals, future research might further investigate the relationship between robot-assisted training and expected clinical improvement. new robot-assisted devices, body-powered robots and their possible advantages (47) or wearable exoskeleton devices that support or replace muscle movement initiation (48) are developed according to new neurophysiological knowledge (49). however, while neurological impairment affects a large number of the population, overall functional recovery of patients with such impairment is limited. as such, further investigation of these available devices, the assessment of their potential for neurologic improvement and any adverse effects is required. acknowledgement. we thank occupational therapists damirka levanić and jurica sinković, who assisted participants in performing the exercises using the exoskeleton device, and to our colleagues, doctors of medicine vesna dragčević, mario fučkar and marija jeršek for making it possible to include patients from their departments in this study. we also thank our institution for the opportunity to use the borrowed armeo®spring hacoma device. disclosure funding. no specific funding was received for this study. transparency declaration: the authors report no 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rehabilitation: a proof of concept study. restor neurol neurosci. 2018;36(6):693708. doi: 10.3233/rnn-180830. 48. mikolajczyk t, klodowski a, mikolajewska e, walkowiak p, berjano p, villafañe jh, aggogeri f, borboni a, fausti d, petrogalli g. design and control of system for elbow rehabilitation: preliminary findings. adv clin exp med 2018; 27(12):1661-1669. doi: 10.17219/acem/74556. 49. gassert r, dietz v. rehabilitation robots for the treatment of sensorimotor deficits: a neurophysiological perspective. journal of seemedj 2021, vol 5, no. 1 upper extremity rehabilitation and neurologic impairment 108 southeastern european medical journal, 2021; 5(1) neuroengineering and rehabilitation. 2018 jun 5;15(1):46. doi: 10.1186/s12984-018-0383-x. 1 author contribution. acquisition of data: blažinčić v, ščurić i, klepo i administrative, technical or logistic support: blažinčić v, ščurić i, klepo i, dubroja i, cerovec d analysis and interpretation of data: blažinčić v, ščurić i, klepo i, dubroja i, cerovec d critical revision of the article for important intellectual content: blažinčić v, ščurić i, klepo i, dubroja i, cerovec d drafting of the article: blažinčić v, ščurić i, klepo i, dubroja i, cerovec d final approval of the article: blažinčić v, ščurić i, klepo i, dubroja i, cerovec d guarantor of the study: blažinčić v, ščurić i, klepo i, dubroja i, cerovec d provision of study materials or patients: blažinčić v, ščurić i, klepo i statistical expertise (statistical analysis of data): blažinčić v, ščurić i, klepo i, dubroja i, cerovec d seemedj 2022, vol 6, no 1 transfusion-transmissible infections in guyana 92 southeastern european medical journal, 2022; 6(1) original article trends in transfusion-transmissible infections among blood donors at the national blood transfusion service, guyana 1 francine leitch 1, letisha pooran 1, rajini kurup 1*, pedro lewis 1, cecil boston 1 1 university of guyana *corresponding author: rajini kurup, rajini.kurup@uog.edu.gy received: feb 14, 2022; revised version accepted: mar 22, 2022; published: apr 27, 2022 keywords: transfusion-transmissible infections, blood bank abstract aim: the most adverse effect of blood transfusion is the acquisition of transfusion-transmissible infections (ttis), which poses a serious threat in developing countries. this study aims to identify the trends of transfusion-transmissible infections among blood donors. materials and methods: this study was a laboratory-based retrospective study conducted using blood donors’ records from january 2015 to december 2018, collected at the national blood transfusion service, guyana (nbts). analysis of data was performed using the statistical package for the social sciences (spss) version 22.0 software and the results were presented in tables and graphs. chi-square and logistic regression were used to identify trends and influencing factors. results: a total of 39,308 blood donors were included in this study, of whom 2,418 (6.2%) donors tested positive to at least one pathogen. among those donors, 4.4% were coinfected with at least one of the sixteen dual infection combinations. the overall seroprevalence of hiv, htlv, syphilis, hbv, hcv, chagas, microfilaria, and malaria was 0.8%, 0.8%, 0.6%, 1.5%, 1.3%, 1.2%, 0.0%, and 0.0%, respectively. trends of transfusion-transmissible infections showed an overall increase from the lowest prevalence, 5.1%, in 2015 to 7% in 2016, followed by decreases in 2017 (6.8%) and 2018 (5.8%). conclusions: even though 98.6% of the donor population are volunteers, this study has shown that a significant percentage of blood donors harbour transfusion-transmissible infections. stringent screening and preventive measures are very important to ensure the safety of the transfusion recipient. (leitch f, pooran l, kurup r, lewis p, boston c. trends in transfusion-transmissible infections among blood donors at the national blood transfusion service, guyana. seemedj 2022; 6(1); 92-104) seemedj 2022, vol 6, no 1 transfusion-transmissible infections in guyana 93 southeastern european medical journal, 2022; 6(1) introduction globally, transfusion of human blood is an essential medical procedure. although it can save lives, blood and/or its products are not 100 percent safe for transfusion. in some cases, there is about one percent (1%) chance of adverse immunological, physiological, and infectious complications in the recipients. of all adverse effects of transfusion, transfusiontransmissible infections (tti) are the most significant and represent a risk for blood safety in developing countries like guyana. ttis include human immunodeficiency virus (hiv), human t-cell lymphotropic virus (htlv), hepatitis b virus (hbv), hepatitis c virus (hcv), malaria parasite (mp), microfilaria parasite (mfp), trypanosoma cruzi (chagas), and syphilis. as a result, the world health organization (who) recommends that all donated blood be tested for ttis caused by these pathogens (1). in addition to serological screening, all campaigners for blood donation should be submitted to clinical screening, which consists of an interview with a trained professional, in order to establish the clinical history and life of the donor concerning exposure to risk factors for ttis. in guyana and other countries around the world, voluntary donors and replacement donors represent the main source of blood for transfusion. for better patient safety, the world health organization (who) recommends that before blood is released for clinical use, it should be screened for evidence of any possible infection. countries with stringent routine serological screening have achieved a very impressive reduction of tti cases. however, the risks persist due to the limited virus detection techniques (2–4). the magnitude of the tti problem varies from country to country; even within a country, the magnitude could vary between different regions depending on the load of transfusion-transmissible infections in that particular population. blood transfusion, in general, is a very expensive process, both as the entire procedure and in case any infection. any infected blood that might reach a patient could lead to morbidity and mortality risks. this could pose an economic burden on the recipients themselves, as well as their families (5). ttis have a higher chance of reaching a wider population since some infections have a long asymptomatic period or some persons could act as the carrier. the costs that would be added to such transmission include an aggressive treatment plan and short or long-term dependency, further burdening the country’s economy (6). viruses like hiv, hbv, and hcv can cause longterm carrier states, prolong viremia and infectivity, lead to chronic disorders with high rates of morbidity and mortality because of chronicity, liver cirrhosis, hepatoma, and other opportunistic infections (7–9). these viruses have a direct transmission route with blood products during transplantation, hemodialysis, intravenous drug use, tattooing as well as sexual intercourse (10). the chance of transmission of those viruses through transfusion of infected blood is higher than with other routes of transmission, mainly due to transmission of a high viral load per transfusion. regardless of whether the viral load is low within the blood, the possibility of infection remains high (9). however, currently, transfusion has a relatively low contribution to the transmission of virus infections since the obligation of screening blood donations for viral infections before transfusion is of the highest priority (2). similarly, parasites are rare, but commonly recognized infectious microbes worldwide, and several protozoans are known to be transmitted through blood transfusion. parasites such as plasmodium spp. (p. falciparum, p. vivax, p. malariae, and p. ovale) and trypanosoma cruzi are causative agents of malaria and chagas disease, respectively (11). the prevalence of these infectious agents among blood donors varies from nation to nation, depending on the specific population from which the blood units are collected. in sub-saharan africa, 5–10% of hiv infections are caused by transfusion of infected blood and 12.5% of patients who receive such blood transfusions are at risk of developing post-transfusion hepatitis (12). the choice of blood donors with low tti risk was followed by seemedj 2022, vol 6, no 1 transfusion-transmissible infections in guyana 94 southeastern european medical journal, 2022; 6(1) effective laboratory screening of blood transfusion units (8, 13). those activities were extremely effective; however, the transmission of diseases nevertheless rose due to the incapacity of laboratory testing to detect the donors with an infection during the window period, a lack of budget for all general laboratories for tti testing and trained manpower, the presence of immunologically variant viruses, the presence of nonseroconverting silent carriers, laboratory testing errors and poor quality control of laboratory tests (14, 15). variations in donor screening strategies and the predominance of risk factors in society might explain the changes in prevalence rates of ttis over time. it is, therefore, necessary to assess the prevalence of these ttis among blood donors at regular intervals to estimate the current most prevalent risk factors and to evaluate the effectiveness of blood safety strategies employed in the blood banks (16). evaluation of trends in the prevalence of ttis among blood donors is not only essential for estimating the effectiveness of blood safety strategies (10, 13); it also provides information to policymakers for the purpose of improving strategies for minimizing the potential risk of acquiring such infections through blood transfusion (17). as a result, to our knowledge, there are no comprehensive data to determine the trends of transfusion-transmissible infections among blood donors at the nbts, guyana. therefore, the primary objective of this study was to determine the prevalence and trends of major ttis among blood donors at the nbts, guyana. subjects and methods the study was carried out at guyana’s main blood bank, the national blood transfusion service (nbts) in georgetown, guyana, by extracting data from the nbts database. nbts is the only blood bank in georgetown, the capital city of guyana. all blood donors are screened for infectious diseases at the nbts. the facility provides tti-tested blood and blood products for many referral hospitals in the region. the center has several departments, sections, and subsections. it comprises the donor clinic, laboratory (sub-sections: tti, immunohematology and component preparation), quality management and data management sections. this research used the retrospective descriptive study method, in which the donor data were collected from the period between january 2015 and december 2018. screening methods the donors’ blood was screened for ttis after donation. blood samples were tested using two kits, based on the recommendation by who, using two different testing strategies involving enzyme-linked immunosorbent assay (elisa) and/or simple or rapid assays for surveillance. cortez diagnostic inc. rpr test was used for screening for syphilis. the positive rpr test was confirmed by a quantitative rpr test. the presence of malaria and microfilaria parasites was established using the peripheral blood smear test. the hepatitis b surface antigen (hbsag) was detected using the murex hbsag confirmatory version 3.0 elisa infectious disease; the kit had a sensitivity of 100% and specificity of 99.97%. antibodies to hcv were detected using the murex anti-hcv version 4.0 elisa infectious disease, which had a sensitivity of 100% and specificity of 99.88%. antibodies to hiv types 1 and 2 were screened using the murex hiv ag/ab combination elisa infectious disease. the kit had a sensitivity of 100% and a specificity of 99.78%. antibodies to htlv types 1 and 2 were screened using the murex htlv i/ii elisa infectious disease. the kit had a sensitivity of 100% and a specificity of 99.88%. antibodies for chagas were screened using the grupobios s.a. test elisa chagas iii. the kit had a sensitivity of 100% and a specificity of 100%. ethical approval the study was approved by the institutional review board (irb) of the ministry of public health, guyana. permission was also obtained from the director of the national blood transfusion service. seemedj 2022, vol 6, no 1 transfusion-transmissible infections in guyana 95 southeastern european medical journal, 2022; 6(1) statistical analysis all data were retrieved from the anonymized blood donor register, donor cards, and logbooks. delphyn blood bank software was used by the principal researchers with the help of the blood bank staff. donors’ records containing specific sociodemographic information (age, sex, ethnicity, donor type, seropositivity for ttis, blood type, and blood donor code), the number of donations and the serostatus of ttis were transferred to an excel spreadsheet (microsoft inc.) and analyzed using spss (statistical package for the social sciences) software version 22.0. table 1: demographic characteristics of blood donors (2015–2018), nbts guyana characteristic n (%) sex male 23766 (60.6) female 15458 (39.4) age (years) 15–20 3652 (9.3) 21–30 12776 (32.5) 31–40 10191 (25.9) 41–50 7728 (19.7) > 51 4958 (12.6) donor type voluntary – 1st time 12211 (31.1) voluntary – regular 26537 (67.5) family replacement –1st time 363 (0.9) family replacement – regular 163 (0.4) ethnicity afro-guyanese 11346 (28.9) indo-guyanese 18407 (46.8) mixed 8482 (21.6) amerindian 615 (1.6) caucasian 187 (0.5) chinese 28 (0.1) others 232 (0.6) blood group (abo + rhesus) a+ 7943 (20.2) b+ 9041 (23.0) o+ 18133 (46.2) ab+ 2106 (5.4) a387 (1.0) b424 (1.1) o1086 (2.8) ab119 (0.3) location inhouse 16909 (43.0) g/town mobile drive 14739 (37.5) region 2 640 (1.6) region 3 948 (2.4) region 6 5047 (12.8) region 9 16 (0.0) region 10 1005 (2.6) seemedj 2022, vol 6, no 1 transfusion-transmissible infections in guyana 96 southeastern european medical journal, 2022; 6(1) the seroprevalence of hiv, htlv, hcv, hbsag, chagas, syphilis, malaria, and microfilaria was expressed in percentages for the entire study group and based on different sociodemographic characteristics (age, sex, ethnicity, region, donor blood type, and donor category) and frequency of donation. descriptive statistics were performed, and the results were presented as percentages in tables and graphs. the chisquared test for trend was applied to examine the variation in trends. logistic regression was used to explore the association between dependent and independent variables. the associations are presented as odds ratios (or), together with 95% confidence intervals (ci). pvalues of less than 0.05 were considered statistically significant. results sociodemographic characteristics of blood donors a total of 39,308 blood donors were screened at the national blood transfusion service (nbts) from january 2015 to december 2018, with 98.6% of those donors being volunteers (table 1 and table 2). of the total, 43% of blood donors donated blood at the central blood bank location. overall, a larger percentage (60.6%) of blood donors were male, while most (32.5%) of the study subjects were in the 21–30 age group. of all donors, 46.2% of them had the o-positive blood type. most of the donor population were indo-guyanese (46.8%) followed by afroguyanese (28.9%) (table 1). table 2. total blood donors, volunteer vs replacement, in the years 2015–2018 at the national blood transfusion service (nbts), guyana. type of donor 2015 2016 2017 2018 p-value vf 3270 (31.6) 6401 (32.6) 2903 (29.7) 2839 (30.3) 0.00 vr 6910 (66.8) 12934 (65.9) 6751 (69.2) 6407 (68.3) 0.00 rf 101 (1.0) 224 (1.1) 69 (0.7) 81 (0.9) 0.003 rr 56 (0.5) 58 (0.3) 29 (0.3) 49 (0.5) 0.001 total 10337 19617 9752 9376 voluntary first time – vf; voluntary repeat – vr; replacement first time – rf; replacement repeat – rr seroprevalence of transfusiontransmissible infections (ttis) a total of 2,418 (6.2%) blood donors tested positive for at least one tti agent. the positivity rates of hiv, htlv, syphilis, hbv, hcv, chagas, microfilaria, and malaria were 0.8%, 0.8%, 0.6%, 1.5%, 1.3%, 1.2%, 0.0%, and 0.0%, respectively. overall, hbv was the most prevalent tti (figure 1). among the 2,418 blood donors, 107 (4.4%) donors were coinfected. sixteen dual infection combinations were observed, with chagas and hcv being the most common (0.7%), followed by hbv–chagas (0.5%), htlv–hbv (0.5%), and hiv– htlv (0.4%) (figure 2). seemedj 2022, vol 6, no 1 transfusion-transmissible infections in guyana 97 southeastern european medical journal, 2022; 6(1) figure 1. prevalence of transfusion-transmissible infections in 2015–2018 at the national blood transfusion service (nbts), guyana. (microf: microfilaria; htlv: human t-lymphotropic virus type 1; hiv: human immunodeficiency virus; hcv: hepatitis c virus; hbv: hepatitis b virus) trends and associated sociodemographic factors of blood donors regarding ttis (2015–2018) the overall trend of ttis showed an increase from the lowest prevalence, 5.1%, in 2015 to 7% in 2016, followed by decreases in 2017 (6.8%) and 2018 (5.8%). hbv was the most prevalent tti (1.5%), with a significant (p ≤ 0.05) progressive increase from 2015 (1.3%) to 2017 (1.8%), followed by a subsequent decrease in 2018 (1.3%) (table 3). overall, in regard to donor types, no tti was significantly prevalent in both first-time and regular family replacement blood donors (p ≥ 0.05). htlv, syphilis, hbv, and microfilaria were significantly (p ≤ 0.05) prevalent in both first-time and regular voluntary donors, while hiv was significantly (p ≤ 0.05) prevalent in just first-time voluntary donors. chagas was not significantly present in any of the blood donor types. htlv (43.9%) and hbv (45.8%) were the significantly prevalent ttis in afro-guyanese donors (p ≤ 0.05), while chagas (37.6%, p ≥ 0.05) was statistically prevalent in indo-guyanese donors. tti positive blood donors with o positive (o+) blood type had a statistically significant prevalence of syphilis (40.9%, p ≤ 0.05). all ttis except hiv, syphilis, and microfilaria showed significant prevalence in blood donors who donated mostly at the blood bank or via mobile blood drives (table 3). 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 microf syphilis htlv hiv hcv hbv chagas 0.01 0.56 0.79 0.86 1.40 1.54 1.15 seemedj 2022, vol 6, no 1 transfusion-transmissible infections in guyana 98 southeastern european medical journal, 2022; 6(1) table 3. reactive serological marker, type of donor, sex, age group at nbts (2015–2018) characteristic n (%) total 2015 2016 2017 2018 p value reactive serological marker hiv 307 (0.8) 75 (0.7) 116 (1.2) 66 (0.7) 50 (0.5) 0.00 htlv 312 (0.8) 68 (0.7) 76 (0.8) 86 (0.9) 82 (0.9) 0.2 syphilis 227 (0.6) 30 (0.3) 51 (0.5) 66 (0.7) 80 (0.9) 0.00 hbv 600 (1.5) 139 (1.3) 158 (1.6) 179 (1.8) 124 (1.3) 0.01 hcv 510 (1.3) 122 (1.2) 180 (1.8) 106 (1.1) 102 (1.1) 0.00 chagas 458 (1.2) 89 (0.9) 108 (1.1) 159 (1.6) 102 (1.1) 0.00 microfilaria 4 (0.0) 1 (0.0) 3 (0.0) 0 0 0.1 malaria 0 0 0 0 0 sex male 23766 (60.6) 6213 (60.1) 5749 (58.8) 5901 (60.6) 5903 (63.0) female 15458 (39.4) 4121 (39.9) 4027 (41.2) 3842 (39.4) 3468 (37.0) 0.00 age group 15–20 3652 (9.3) 1098 (10.6) 982 (10.0) 839 (8.6) 733 (7.8) 21–30 12776 (32.5) 3263 (31.5) 3407 (34.7) 3080 (31.6) 3026 (32.3) 31–40 10191 (25.9) 2632 (25.4) 2384 (24.3) 2614 (26.8) 2561 (27.3) 41–50 7728 (19.7) 2075 (20.0) 1879 (19.1) 1936 (19.8) 1838 (19.6) > 51 4958 (12.6) 1283 (12.4) 1164 (11.9) 1292 (13.2) 1219 (13.0) 0.00 type of donor voluntary – 1st time 12211 (31.1) 3270 (31.6) 3199 (32.6) 2903 (29.7) 2839 (30.3) 0.00 voluntary – regular 26537 (67.5) 6910 (66.8) 6468 (65.9) 6752 (69.2) 6407 (68.3) 0.00 family replacement – 1st time 363 (0.9) 101 (1.0) 112 (1.1) 69 (0.7) 81 (0.9) 0.01 family replacement – regular 163 (0.4) 56 (0.5) 29 (0.3) 29 (0.3) 49 (0.5) 0.004 htlv was significantly (p ≤ 0.05) more prevalent among female donors (58%) compared to hbv, which was significantly (p ≤ 0.05) more prevalent in males (65.7%). a significant prevalence of ttis – hbv (35.7%), hcv (31.2%), and chagas (37.8%) – was seen especially in the 21–30 age group, while htlv (26.9%) was significantly observed in the > 51 age group (p ≤ 0.05) compared to other age groups (table 4). overall, in regard to donor types, no tti was significantly prevalent in both first-time and regular family replacement blood donors (p ≥ 0.05). htlv, syphilis, hbv, and microfilaria were significantly (p ≤ 0.05) prevalent in both first-time and regular voluntary donors, while hiv was significantly (p ≤ 0.05) prevalent in just first-time voluntary donors. chagas was not significantly present in any of the blood donor types. htlv (43.9%) and hbv (45.8%) were the significantly prevalent ttis in afro-guyanese donors (p ≤ 0.05), while chagas (37.6%, p ≥ 0.05) was statistically prevalent in indo-guyanese donors. tti positive blood donors with o positive (o+) blood type had a statistically significant prevalence of syphilis seemedj 2022, vol 6, no 1 transfusion-transmissible infections in guyana 99 southeastern european medical journal, 2022; 6(1) (40.9%, p ≤ 0.05). all ttis except hiv, syphilis, and microfilaria showed significant prevalence in blood donors who donated mostly at the blood bank or via mobile blood drives (table 4). table 4. blood donor characteristics and chi-square for transfusion-transmissible infections (ttis), 2015–2018, nbts, guyana. characteristic hiv (307) htlv (312) syphilis (227) hbv (600) hcv (510) chagas (458) microfilaria (4) sex male 185 (60.3) 131 (42.0) 145 (64.2) 394 (65.7) 317 (62.2) 274 (60.0) 1 (25.0) female 122 (39.7) 181 (58.0) 81 (35.8) 206 (34.3) 193 (37.8) 183 (40.0) 3 (75.0) χ2 0.1 45.6 1.2 6.6 0.5 0.08 2.1 p value 0.9 0.00 0.3 0.01 0.5 0.80 0.1 age (years) 15–20 28 (9.1) 17 (5.4) 17 (7.5) 58 (9.7) 36 (7.1) 66 (14.4) 0 21–30 104 (33.9) 78 (25.0) 80 (35.4) 214 (35.7) 159 (31.2) 173 (37.8) 3 (75.0) 31–40 63 (20.5) 73 (23.4) 54 (23.9) 185 (30.8) 103 (20.2) 96 (21.0) 0 41–50 76 (24.8) 60 (19.2) 41 (18.1) 95 (15.8) 122 (23.9) 87 (19.0) 1 (25.0) > 51 36 (11.7) 84 (26.9) 34 (15.0) 48 (8.0) 90 (17.6) 36 (7.9) 0 χ2 7.9 62.3 3 22.4 24.7 29.8 4.2 p value 0.1 0.00 0.6 0.00 0.00 0.00 0.4 donor type voluntary – 1st time 113 (36.8) 121 (38.8) 92 (40.5) 244 (40.7) 172 (33.7) 159 (34.7) 4 (100.0) χ2 4.6 8.7 9.5 26.2 1.7 2.9 8.9 p value 0.03 0.003 0.002 0.00 0.2 0.09 0.00 voluntary – regular 192 (62.5) 185 (59.3) 132 (58.1) 346 (57.7) 335 (65.7) 294 (64.2) 0 χ2 3.5 9.7 9.1 26.9 0.8 2.3 8 p value 0.06 0.002 0.003 0.00 0.4 0.1 0.00 family replacement – 1st time 0 4 (1.3) 3 (1.3) 8 (1.3) 2 (0.4) 3 (0.7) 0 χ2 4.1 0.4 0.4 1.1 1.6 0.4 0.04 p value 0.04 0.5 0.5 0.3 0.2 0.5 0.8 family replacement – regular 0 2 (0.6) 0 1 (0.2) 2 (0.4) 1 (0.2) 0 χ2 1.2 0.4 0.9 0.9 0.006 0.4 0.03 p value 0.3 0.5 0.3 0.3 0.9 0.5 0.8 ethnicity afro-guyanese 89 (29.0) 137 (43.9) 81 (35.7) 275 (45.8) 142 (27.8) 153 (33.4) 2 (50.0) indo-guyanese 142 (46.3) 77 (24.7) 105 (46.3) 196 (32.7) 253 (49.6) 172 (37.6) 1 (25.0) mixed 74 (24.1) 84 (26.9) 36 (15.9) 122 (20.3) 102 (20.0) 118 (25.8) 1 (25.0) amerindians 1 (0.3) 12 (3.8) 3 (1.3) 6 (1.0) 10 (2.0) 10 (2.2) 0 caucasians 0 1 (0.3) 0 0 2 (0.4) 3 (0.7) 0 seemedj 2022, vol 6, no 1 transfusion-transmissible infections in guyana 100 southeastern european medical journal, 2022; 6(1) others 1 (0.3) 1 (0.3) 2 (0.9) 0 1 (0.2) 2 (0.4) 0 χ2 6.0 72.9 8.8 93.7 3.9 17.5 1.2 p value 0.40 0.00 0.2 0.00 0.7 0.01 0.9 blood group a+ 59 (19.2) 79 (25.4) 55 (24.4) 117 (19.6) 114 (22.4) 88 (19.2) 1 (25.0) a2 (0.7) 1 (0.3) 3 (1.3) 3 (0.5) 5 (1.0) 4 (0.9) 0 b+ 72 (23.5) 72 (23.2) 51 (22.7) 135 (22.6) 100 (19.6) 112 (24.5) 1 (25.0) b7 (2.3) 1 (0.3) 0 4 (0.7) 6 (1.2) 3 (0.7) 0 o+ 136 (44.3) 131 (42.1) 92 (40.9) 297 (49.7) 243 (47.6) 218 (47.6) 2 (50.0) o15 (4.9) 9 (2.9) 7 (3.1) 15 (2.5) 8 (1.6) 12 (2.6) 0 ab+ 15 (4.9) 15 (4.8) 12 (5.3) 26 (4.4) 32 (6.3) 19 (4.1) 0 ab1 (0.3) 3 (1.0) 5 (2.2) 0 2 (0.4) 2 (0.4) 0 χ2 10.1 13.0 33.7 7.3 7.6 3.3 0.5 p value 0.2 0.07 0.00 0.40 0.40 0.90 1 location inhouse 118 (38.4) 96 (30.8) 100 (44.1) 198 (33.0) 207 (40.6) 142 (31.0) 1 (25.0) g/town mobile drive 136 (44.3) 145 (46.5) 82 (36.1) 282 (47.0) 185 (36.3) 241 (52.6) 3 (75.0) region 2 4 (1.3) 11 (3.5) 4 (1.8) 13 (2.2) 18 (3.5) 7 (1.5) region 3 10 (3.3) 13 (4.2) 13 (5.7) 20 (3.3) 8 (1.6) 9 (2.0) region 6 36 (11.7) 35 (11.2) 26 (11.5) 73 (12.2) 83 (16.3) 48 (10.5) region 9 0 0 0 0 0 0 region 10 3 (1.0) 12 (3.8) 2 (0.9) 14 (2.3) 9 (1.8) 11 (2.4) χ2 9.9 31.5 13.6 32.7 20.2 46.5 2.6 p value 0.2 0.00 0.06 0.00 0.005 0.00 0.9 logistic regression analysis shows that male donors have a significant (or = 0.57, ci: 0.45–0.71) chance of presenting with htlv and hbv, with odds of 0.51 and 1.51, respectively, when compared to female donors. additionally, blood donors in the 41–50 age group compared to those in the > 51 age group, with blood types o+, a-, and b-, compared to ab-, had significant (p ≤ 0.05) risk of presenting with htlv. blood donors who donated in region 2 were at a significantly higher risk of htlv and hcv infection compared to those in region 10. furthermore, logistic regression analysis showed that the odds of blood donors presenting with syphilis significantly (p ≤ 0.05) increased with age, while all age groups except 31–40 (p ≥ 0.05) had a significant chance of presenting with chagas. discussion over the observed four years, this study showed that 2,418 donors tested positive for at least one of the eight tti markers that were screened. the study had similar findings as observed in the studies conducted in eastern ethiopia by teklemeriam et al (18) and by birhaneselassi et al (19). our findings included a higher percentage compared to similar studies conducted in the neighboring brazil (20), in south india (21), in andaman and nicobar islands (22), in south gujarat, india (23), in kano, nigeria (8) and in south-south nigeria (24). the differences in prevalence among these studies may be due to the existence of different magnitudes of risk factors for contracting transfusion-transmissible infections, seemedj 2022, vol 6, no 1 transfusion-transmissible infections in guyana 101 southeastern european medical journal, 2022; 6(1) discrepancies in the overall sample size, donor enrolment, the observed period, as well as factors associated with testing procedures used for screening, storage, and validation of test kits. of the eight ttis that were studied, the most common was hbv, followed by hcv, chagas, hiv, htlv, syphilis, and malaria. the rate of tti variation depends on improvement in analytical technology, which could improve the current screening reagents, not only to make them more specific, but also more reliable (25). likewise, our study revealed that hbv was significantly more prevalent among males when compared to female donors; this might be due to the fact that sex is a genetic factor of disease consequence. concerning hbv infection, when this virus infects an adult, most subjects will produce protective antibodies and fully fight off the infection. but in a few subjects (5%–10%), the virus will establish a chronic infection. moreover, because of the slow plasma disappearance rate for hbsag in males compared to females, males are more likely to develop chronic hbv infection. in addition to the above-mentioned reasons, behavioral risk factors such as having multiple sex partners could be the cause of the increase in prevalence of hbv among male donors (26). hiv’s seroprevalence in this study is lower compared to a previous study performed at the same blood bank during 2010–2011 (1.42%) (27) and the 2016 national estimated hiv prevalence (1.6%) (28). many factors could be the cause of the differences in seroprevalence recorded in some developing nations, such as cultural and religious differences, differences in education level, and different socioeconomic structure (8, 24). having multiple or concurrent sex partners, dependence on unskilled persons for childbirth, instruments used for female genital mutilation, cultural tattooing or markings on the body are some of the sociocultural and religious practices involved in the risk of transmission (29). a lot of effort needs to be put in to reduce such practices and to prevent the spread of infections. introducing aggressive campaigns and counseling before and after blood transfusions might in some way help reduce transmission (2). this study shows an increasing trend in blood donations testing positive for ttis in the last four years. one of the reasons associated with the increasing trend of ttis in donated blood during 2016 could be the increased number of first-time blood donors in that particular year, since studies have identified that first-time blood donors always tend to pose a greater risk of infectious donation than repeat donors. one assumption is that repeat donors are wellinformed of the risky behaviors that might cause blood infections and as such reduce the probability of blood infections in the window period. furthermore, if a donor tests negative prior to a donation, it is likely that most repeat donors will not suddenly engage in high-risk behaviors. therefore, it is very important to ensure the safety of blood supply through careful recruitment of new donors and maintaining the donation pool (2). this study had a few limitations. many epidemiological data were not collected since this was a retrospective study. there could be much more information if it is collected with the aim of better establishing the risks of tti infection. this study did not do any confirmatory tests nor were any molecular data evaluations done. repeated positive tests were considered positive for the purposes of this study. as such, false positive cases cannot be completely excluded. despite the limitations, this study highlighted the trends and associated sociodemographic factors of major ttis. acknowledgement. we extend our gratitude to the technologists, technicians, and staff of the national blood transfusion service (nbts) for their assistance during this research work. disclosure funding. no specific funding was received for this study competing interests. none to declare. seemedj 2022, vol 6, no 1 transfusion-transmissible infections in guyana 102 southeastern european medical journal, 2022; 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northwest ethiopia. bmc infect dis. 2019; 19(1):393. doi: 10.1186/s12879-019-3950-2. 26. shiferaw e, tadilo w, melkie i, shiferaw m. sero-prevalence and trends of transfusiontransmissible infections among blood donors at bahir dar district blood bank, northwest ethiopia: a four year retrospective study. plos one. 2019; 14(4):e0214755. doi: 10.1371/journal.pone.0214755. 27. kurup r, barclay c. a study on the prevalence of hiv among blood donors in guyana. j dis glob heal 2015; 3(1):39–43. retrieved from https://www.ikprress.org/index.php/jodagh/ article/view/503 28. center for disease control and prevention (cdc). global hiv and tb. 2016. available from: https://www.cdc.gov/globalhivtb/where-wework/guyana/guyana.html 29. bharti ks, lodha nd. changing trends of transfusion transmissible infections in blood donors in vidharbha region: a retrospective study of thirteen years. international journal of contemporary medical research 2018;5(9):i5-i9.. seemedj 2022, vol 6, no 1 transfusion-transmissible infections in guyana 104 southeastern european medical journal, 2022; 6(1) doi: http://dx.doi.org/10.21276/ijcmr.2018.5.9.23 author contribution. acquisition of data: fl, lp, rk, pl, cb administrative, technical or logistic support: fl, lp, rk, pl, cb analysis and interpretation of data: fl, lp, rk, pl, cb conception and design: fl, lp, rk, pl, cb critical revision of the article for important intellectual content: fl, lp, rk, pl, cb drafting of the article: fl, lp, rk, pl, cb final approval of the article: fl, lp, rk, pl, cb provision of study materials or patients: fl, lp, rk, pl, cbstatistical expertise: fl, lp, rk, pl, cb seehsj south eastern europe health sciences journal southeastern european medical journal seemedj faculty of medicine osijek josip juraj strossmayer university of osijek j. huttlera 4 croatia – 31000 osijek seemedj@mefos.unios.hr seemedj.mefos.unios.hr dear colleagues, hereby, new 10th issue of seemedj is presented to you. this issue brings flaming topic of covid-19 disease and some intriguing questions on transmission and mortality of covid-19 in cancer patients in relation to blood group (ebinc et al). article by bogović et al reviewed clinical outcomes of covid-19. anaemia of chronic disease is more common in covid-19 positive patients and the clinical outcomes of covid-19 disease is poorer. also, an advanced inflammatory condition characterized with higher ferritin/transferrin ratio may be a predictive factor of intensive care unit admission in these patients. in line with wide discussion on pro and contra vaccination, article by smajić et al. present a survey on university students and their attitudes towards responsible behavior and vaccination, finding interesting differences in favor to students form biomedical fields. work in shifts significantly affects arterial blood pressure and higher ferritin levels in women, suggesting subclinical inflammatory process, possibly important in etiology of hypertension (cvitkušić-lukenda et al). furthermore, pavličević tomas and degmečić review data of 800 patients in psychiatry clinic and found out that almost one third of them suffered from hypertension which was related to recurrent depressive disorder, alcohol addiction and posttraumatic stress disorder. outcomes of deep vein thrombosis in emergency department with emphasis on treatments with novel oral anticoagulants (noac) is presented in article by radilj et al. while chronic kidney disease related anemia is reviewed in study by hrvačić et al. and finally, importance of effective minimization of blood usage and wastage in guyana is presented in article by kurupa et al. the art work at the cover page is a sculpture from the first half of 20th century, a work by mihajlo živić, artist from osijek, whose work was influenced by ivan meštrović. i hope that readers will find relevant published articles for their work on a behalf of editorial board and my own, i warmly greet our readers and invite them to join us in the endeavor of publishing own scientific work in seemedj. ines drenjančević, md, phd editor-in-chief southeastern european medical journal (seemedj) seemedj 2022, vol 6, no 1 hypertension and covid-19 outcomes 21 southeastern european medical journal, 2022; 6(1) original article association between common comorbidities and outcomes in covid-19 patients hospitalised in general hospital našice – a crosssectional study 1 lea gvozdanović 1, željka dragila 2, zvjezdana gvozdanović 1,3, denis klapan 1,3, nikolina farčić 2,3,4, hrvoje šimić 1,3, zrinka mihaljević 4* 1 general hospital našice, croatia 2 university hospital centre osijek, croatia 3 faculty of dental medicine and health osijek, j. j. strossmayer university of osijek, croatia 4 faculty of medicine osijek, j. j. strossmayer university of osijek, croatia *corresponding author: zrinka mihaljević, zrinka.mihaljevic@mefos.hr received: mar 2, 2022; revised version accepted: apr 5, 2022; published: apr 27, 2022 keywords: covid-19, comorbidity, hypertension, mortality abstract aim: the aim was to define the impact of comorbidities, specifically hypertension as one of the most common chronic diseases, on the outcome and length of stay for covid-19 patients. methods: the cross-sectional study, carried out from october to december 2021, included 129 hospitalised covid-19 patients who presented to the emergency department and were hospitalised and treated in the covid ward in the general hospital našice. all patients tested positive for covid19 with a polymerase chain reaction (pcr) test. clinical parameters were also recorded and they included demographic factors, comorbidities, type of antihypertensive therapy, new-onset hypertension, length of stay and the overall outcome. results: the most common comorbidity was hypertension (86, 66.7%). hypertension was associated with women (p = 0.03) and age over 65 years (p < 0.001). length of stay was longer for patients with hypertension (p = 0.04) and/or diabetes mellitus (p = 0.04). higher mortality was associated with age over 65 years (p < 0.001) and a higher number of comorbidities (p = 0.01). new-onset hypertension was recorded in three patients. there was no significant difference in the outcome in relation to antihypertensive therapy. conclusion: hypertension is the most common comorbidity in hospitalised covid-19 patients. although treated hypertension did not have a negative impact on the outcome, other potential risk factors, including a higher number of comorbidities and older age, are associated with mortality in covid-19 patients. (gvozdanović l, dragila ž, gvozdanović z, klapan d, farčić n, šimić h, mihaljević z. association between common comorbidities and outcomes in covid-19 patients hospitalised in general hospital našice – a cross-sectional study. seemedj 2022; 6(1); 21-30) seemedj 2022, vol 6, no 1 hypertension and covid-19 outcomes 22 southeastern european medical journal, 2022; 6(1) introduction coronavirus disease 2019 (covid-19) is an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov2), which causes pneumonia and flu-like symptoms. the first case was reported in wuhan, china in late 2019, after which the virus has spread around the world. shortly afterwards, the coronavirus pandemic has been declared. since the first confirmed case of covid-19, there have been approximately 418,650,474 confirmed cases worldwide, including 5,856,224 deaths, as reported by the who (1). the first case of covid-19 in croatia was reported in february 2020 in zagreb. at the time of writing, there have been approximately 1,041,212 confirmed cases, 14,815 of which were fatal (2). several variants of sars-cov-2 with the far more dominant alpha strain were recorded in early may 2021. in june, the delta strain began to spread more rapidly, and by early september, it was responsible for more than 99% of all covid19 cases. in december 2021, the omicron variant began to break through within the delta strain and a few weeks later, it became the dominant strain (3). although the prognosis for patients with covid19 is generally good, it has been observed that some patients with comorbidities have a poor outcome (4,5). previous studies on covid-19 have reported that over 60% of covid-19 patients have associated comorbidities (4,6). the most common comorbidities are hypertension (7–9), diabetes mellitus (dm) (8,10), cardiovascular diseases (10), chronic obstructive pulmonary disease (7), chronic kidney disease (11) and malignancy (12), which are associated with severe disease and mortality. however, there is conflicting data in this regard. a poor outcome in covid-19 patients is also associated with a higher number of comorbidities (8). it has been observed that three or more comorbidities are the cut-off point for severe covid-19 cases, while four or more comorbidities indicate a high rate of fatal outcomes (4). older age is an independent risk factor for disease progression and higher mortality rates (13). it has been observed that many patients with rapid disease progression are 45-65 years old, while severe disease and most deaths were reported in patients aged 70 and above (14). in general, both age and the number of comorbidities could have a predictive role in disease severity and in the final outcome of covid-19. the length of stay for covid-19 patients depends on several factors. analyses have shown that age, gender, hypertension, dm, cardiovascular disease and renal failure are significantly associated with the length of stay (15). it has been observed that every additional comorbidity was significantly associated with an increase in the expected length of stay by 2%. similarly, every one-year increase in age is also significantly associated with an increase in the expected length of stay by 2% (16). hypertension is one of the most common chronic diseases associated with hospitalised patients, but its impact on covid-19 patients has not been well-defined (17). previous studies indicate that hypertension is associated with a higher risk of all-cause mortality regardless of age, gender and other comorbidities (9). it is also assumed that antihypertensive drugs play a major role in the mechanism of interaction between the coronavirus and target cells in the body through the angiotensin-converting enzyme 2 (ace2) (18,19). this hypothesis has come to light when the following two facts were related: the finding that the renin-angiotensinaldosterone system (raas) inhibitors increase the expression of ace2 and the observation that hypertension and diabetes were the most common comorbidity among patients with severe covid-19, for which raas inhibitors are widely used (20). although the treatment of hypertension with these drugs is thought to increase the risk of developing severe and fatal covid-19, studies have found no such association so far (20–22). the covid-19 pandemic is a global health issue that has spread rapidly around the world with varying degrees of severity. this study aimed to define the significance of comorbidities in seemedj 2022, vol 6, no 1 hypertension and covid-19 outcomes 23 southeastern european medical journal, 2022; 6(1) hospitalised covid-19 patients, specifically hypertension as one of the common chronic diseases worldwide. understanding these factors is key to improve the accuracy of predicting disease outcomes as well as the length of stay for the purpose of optimising resources and providing better care for patients. patients and methods this cross-sectional study, carried out from 1 october to 31 december 2021, included 129 hospitalised covid-19 patients who presented to the emergency department in the general hospital našice, which has been a designated hospital for treating covid-19 patients since november 2020. inclusion criteria were as follows: patients admitted from the emergency department, hospitalised and treated in the covid ward, who tested positive for sars-cov2 with a nasal swab polymerase chain reaction (pcr) test (23) during the admission. exclusion criteria included the following: eight patients transferred from the covid ward to the university hospital centre osijek during hospitalisation. the data were reviewed based on the electronic medical records kept by the emergency department. clinical parameters were also recorded, including demographic factors (age and gender), comorbidities (hypertension, dm, atrial fibrillation and respiratory comorbidities – asthma or chronic obstructive pulmonary disease), type of antihypertensive therapy (angiotensin-converting enzyme (ace) inhibitors, angiotensin receptor blockers (arbs), calcium channel blockers and diuretics), length of stay and outcome (discharge or fatal outcome). with regard to the number of comorbidities, the patients were divided into five groups (0, 1, 2, 3 and 4 comorbidities). depending on the combination of antihypertensive therapy, the patients were divided into four groups (1, 2, 3 and 4 types of antihypertensives). new-onset hypertension was defined as hypertension in patients who did not have hypertension as a comorbidity or who did not have any antihypertensive treatment during admission, but received hypertensive therapy at discharge. regarding age, two groups of patients were defined (<65 and ≥65 years old). in terms of the length of stay, the patients were divided into two groups based on the number of days spent in the covid ward (≤10 or >10 days). the study was approved by the ethics committee of the general hospital našice (official gazette 100/18, 147/2020). statistical analysis categorical and numerical data were used for the statistical analysis. categorical data were presented as absolute and relative frequencies. numerical data were presented in the form of an arithmetic mean and standard deviation in case of normal distribution, and as a median and interquartile range for data not following the normal distribution. differences in categorical variables were tested by the χ2-test. the student’s t-test for independent values was used in case of normal distribution, while the mann–whitney u test and the kruskal–wallis test were used in case of deviations from the normal distribution to test the numerical data. all p-values were two-tailed. the significance level was set to alpha = 0.05. the statistical analysis was performed using the medcalc statistical software, version 20.027 (medcalc software ltd, ostend, belgium, https://www.medcalc.org/, 2022). results the study included 129 patients, of whom 61 (47.3%) were male and 68 (52.7%) were female. the median age was 70 years (interquartile range from 60 to 83 years), while 46 (35.7%) patients were under 65 years of age. the most common comorbidity was hypertension (86, 66.7%), followed by diabetes mellitus (36, 27.9%) and atrial fibrillation (27, 20.9%), whereas only 14 patients (10.9%) had no comorbidities. the diagnosis of hypertension was significantly associated with gender – 75% of women had hypertension, in contrast to 57.4% of men (p = 0.03, χ2 test), and with age – 80.7% of the patients aged 65 and above had hypertension (p < 0.001, χ2 test) (table 1). seemedj 2022, vol 6, no 1 hypertension and covid-19 outcomes 24 southeastern european medical journal, 2022; 6(1) table 1. demographic findings in relation to presence of hypertension hypertension n (%) p* total n (%) yes no gender male 35 (57.4) 26 (42.6) 0.03 61 (47.3) female 51 (75) 17 (25) 68 (52.7) age < 65 19 (41.3) 27 (58.7) <0.001 46 (35.7) ≥ 65 67 (80.7) 16 (19.3) 83 (64.3) total n (%) 86 (66.7) 43 (33.3) 129 (100) * χ2 test the patients stayed in the hospital for a median of 9 days (6 – 12 days). the majority of patients were discharged from the hospital (94, 72.9%), whereas 35 patients (27.1%) had a fatal outcome. a total of 83 patients (64.3%) stayed in the hospital less than 10 days, while 46 (35.7%) stayed longer. the length of stay was significantly associated with the presence of certain comorbidities. in particular, patients with hypertension (p = 0.04, χ2 test) and diabetes mellitus (p = 0.04, χ2 test) had a significantly longer stay. healthy patients had a shorter length of stay, but there was no statistical significance (table 2). table 2. length of stay in relation to different comorbidities length of stay (days) n (%) p* total n (%) ≤10 >10 comorbidity none yes 11 (78.6) 3 (21.4) 0.24 14 (10.9) no 72 (62.6) 43 (37.4) 115 (89.1) hypertension yes 50 (58.1) 36 (41.9) 0.04 86 (66.7) no 33 (76.7) 10 (23.3) 43 (33.3) diabetes mellitus yes 18 (50) 18 (50) 0.04 36 (27.9) no 65 (69.9) 28 (30.1) 93 (72.1) respiratory† yes 6 (60) 4 (40) 0.77 10 (7.8) no 77 (64.7) 42 (35.3) 119 (92.2) atrial fibrillation yes 17 (63) 10 (37) 0.87 27 (20.9) no 66 (64.7) 36 (35.3) 102 (79.1) total n (%) 83 (64.3) 46 (35.7) 129 (100) * χ2 test; † asthma and chronic obstructive pulmonary disease the majority of patients used a combination of two antihypertensive drugs (34, 28.3%), the most common of which were ace inhibitors (55, 45.8%) and diuretics (43, 35.8%). there was no significant difference in the outcome with regard to the type of antihypertensive therapy and the number of antihypertensives used (table 3). new-onset hypertension was detected in three (2.3%) patients. all three patients were under 65 years of age and were discharged. the patients with new-onset hypertension had a shorter mean length of stay. seemedj 2022, vol 6, no 1 hypertension and covid-19 outcomes 25 southeastern european medical journal, 2022; 6(1) table 3. antihypertensive therapy in relation to outcome outcome n (%) p* total n (%) discharge fatal antihypertensive therapy none yes 33 (78.6) 9 (21.4) 0.42 42 (35) no 56 (71.8) 22 (28.2) 78 (65) ace inhibitor yes 41 (74.5) 14 (25.5) 0.93 55 (45.8) no 48 (73.8) 17 (26.2) 65 (54.2) arb yes 5 (62.5) 3 (37.5) 0.43 8 (6.7) no 84 (75) 28 (25) 112 (93.3) beta blocker yes 24 (70.6) 10 (29.4) 0.58 34 (28.3) no 65 (75.6) 21 (24.4) 86 (71.7) calcium channel blocker yes 21 (70) 9 (30) 0.55 30 (25) no 68 (75.6) 22 (24.4) 90 (75) diuretic yes 29 (67.4) 14 (32.6) 0.21 43 (35.8) no 60 (77.9) 17 (22.1) 77 (64.2) combination of antihypertensives 0 33 (78.6) 9 (21.4) 0.16† 42 (35) 1 11 (68.7) 5 (31.2) 16 (13.3) 2 26 (76.5) 8 (23.5) 34 (28.3) 3 19 (73.1) 7 (26.9) 26 (21.7) 4 0 2 (100) 2 (1.7) total n (%) 94 (72.9) 35 (27.1) 129 (100) ace = angiotensin-converting enzyme; arb = angiotensin receptor blocker; * χ2 test; † kruskal–wallis test patients aged 65 and above had significantly higher mortality rates (p <0.001, χ2 test), while gender-related differences in the outcome were not significant. there was no significant association between the investigated comorbidities and outcomes. out of the 14 healthy patients, 1 (7.1%) had a fatal outcome. regarding the hypertensive patient population, 61 patients (70.9%) were discharged and 25 (29.1%) had a fatal outcome. there was a significant association between the number of comorbidities and fatal outcome (p = 0.01, kruskal–wallis test), with patients with one and three comorbidities having the worst outcome (table 4). in addition, the patients with a fatal outcome had a significantly shorter length of stay (median of 7 days), in contrast to discharged patients, whose length of stay was 10 days (p< 0.001, mann–whitney u test). seemedj 2022, vol 6, no 1 hypertension and covid-19 outcomes 26 southeastern european medical journal, 2022; 6(1) table 4. demographic findings, presence and number of comorbidities in relation to outcome outcome n (%) p* total n (%) discharge fatal gender male 44 (72.1) 17 (27.9) 0.86 61 (47.3) female 50 (73.5) 18 (26.5) 68 (52.7) age < 65 42 (91.3) 4 (8.7) <0.001 46 (35.7) ≥ 65 52 (62.7) 31 (37.3) 83 (64.3) comorbidity none yes 13 (92.9) 1 (7.1) 0.08 14 (10.9) no 81 (70.4) 34 (29.6) 115 (89.1) hypertension yes 61 (70.9) 25 (29.1) 0.49 86 (66.7) no 33 (76.7) 10 (23.3) 43 (33.3) diabetes mellitus yes 25 (69.4) 11 (30.6) 0.59 36 (27.9) no 69 (74.2) 24 (25.8) 93 (72.1) respiratory‡ yes 8 (80) 2 (20) 0.6 10 (7.8) no 86 (72.3) 33 (27.7) 119 (92.2) atrial fibrillation yes 18 (66.7) 9 (33.3) 0.42 27 (20.9) no 76 (74.5) 26 (25.5) 102 (79.1) number of comorbidities 0 13 (92.9) 1 (7.1) 0.01† 14 (10.9) 1 27 (62.8) 16 (37.2) 43 (33.3) 2 45 (80.4) 11 (19.6) 56 (43.4) 3 6 (46.2) 7 (53.8) 13 (10.1) 4 3 (100) 0 3 (2.3) total n (%) 94 (72.9) 35 (27.1) 129 (100) * χ2 test; † kruskal–wallis test; difference between 0 and 2 to 1 and 3 comorbidities (post hoc conover); ‡ asthma and chronic obstructive pulmonary disease discussion this research studied the demographic characteristics and comorbidities in covid-19 patients hospitalised in the general hospital našice from 1 october to 31 december 2021, with a focus on the hypertensive population, in relation to the length of stay and outcome. of 129 hospitalised covid-19 patients included in the study, hypertension was identified as the most common chronic disease, followed by diabetes mellitus and atrial fibrillation, which is consistent with previous studies (6,7,10,11,17,24), including studies in croatia (25–27). in croatia, the prevalence of hypertension is high, namely 37% (m – 35.2%, f – 39.7%) according to the 2005 eh-uh study (28). accordingly, in this study, hypertension was significantly more common in females, which is consistent with research conducted in china (17) and the united states (16). hospitalized covid19 patients with hypertension were also significantly older than those without hypertension, which is consistent with research conducted in iran (6). the patients stayed in the hospital for a median of 9 days (6 – 12 days). the study found that comorbidities played a significant role in the length of stay. it was observed that hypertension and diabetes mellitus were more associated with a longer seemedj 2022, vol 6, no 1 hypertension and covid-19 outcomes 27 southeastern european medical journal, 2022; 6(1) stay than other detected comorbidities, corresponding with research conducted in the united states (16) and ghana (15). in this study, the majority of hospitalised covid19 patients with hypertension were treated with ace inhibitors (55, 45.8%), but there was no significant association between the type of antihypertensive therapy and the outcome, which is consistent with previous studies (20–22). since ace2 plays a negative role in the raas system, a decrease in ace2 and an increase in ang2 levels can lead to higher blood pressure values (29). in this study, new-onset hypertension was detected in three (2.3%) patients. all three patients were under 65 years of age and were discharged. in the study conducted in turkey (30), of 153 subjects, newonset hypertension was observed in 18 (12%) patients, which is comparable to our study. there are other potential risk factors, including age and the number of comorbidities, that could lead to more severe disease and increased mortality rates in hospitalised covid-19 patients. the association between a fatal outcome and specific comorbidities was not determined in this study. however, a significant association was detected between a higher number of comorbidities and older age, which is consistent with previous research (7,24,31,32), including studies in croatia (25,27). this study identified hypertension as the most common comorbidity in hospitalized covid-19 patients. even though treated hypertension did not affect the outcome negatively, other potential factors, including the number of comorbidities and age, were shown to be associated with mortality among covid-19 patients. further studies of the mechanism in hypertensive covid-19 population are needed. study limitations this study has a few limitations. first, only 129 subjects with confirmed covid-19 were included in the study and more extensive research would help gain a better understanding of the role of hypertension in covid-19 patients. second, the study was conducted in a short period of time without a control group. finally, the data on outcomes in a short-term and long-term follow-up period after covid-19 are limited. acknowledgement. none. disclosure funding. no specific funding was received for this study competing interests. none to declare. references 1. world health organization. who coronavirus (covid-19) dashboard. https://covid19.who.int/ (accessed 19.02.2022.). 2. johns hopkins coronavirus resource center. https://coronavirus.jhu.edu/region/croatia (accessed 19.02.2022.). 3. from delta to omicron, here’s how scientists know which coronavirus variants are circulating in the us. https://theconversation.com/from-delta-toomicron-heres-how-scientists-know-whichcoronavirus-variants-are-circulating-in-the-us173971 (accessed19.02.2022.). 4. liu d, zheng y, kang j, wang d, bai l, mao y, zha g, tang h, zhang r. not only high number and specific comorbidities but also age are closely related to progression and poor prognosis in patients with covid-19. front med (lausanne). 2022; 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75(7):1730-1741. doi: 10.1111/all.14238. 32. lippi g, wong j, henry bm. hypertension in patients with coronavirus disease 2019 (covid-19): a pooled analysis. pol arch intern seemedj 2022, vol 6, no 1 hypertension and covid-19 outcomes 30 southeastern european medical journal, 2022; 6(1) med. 2020; 130(4):304-309. doi: 10.20452/pamw.15272. 1 author contribution. acquisition of data: gvozdanović l, klapan d administrative, technical or logistic support: gvozdanović l, dragila ž, gvozdanović z, klapan d, farčić n, šimić h, mihaljević z analysis and interpretation of data: gvozdanović l, dragila ž, klapan d, mihaljević z conception and design: gvozdanović l, klapan d, mihaljević z critical revision of the article for important intellectual content: gvozdanović l, dragila ž, gvozdanović z, klapan d, farčić n, šimić h, mihaljević z drafting of the article: gvozdanović l, klapan d, mihaljević z final approval of the article: gvozdanović l, dragila ž, gvozdanović z, klapan d, farčić n, šimić h, mihaljević z statistical expertise: dragila ž seemedj 2021, vol 5, no. 1 factors impacting phase angle values 89 southeastern european medical journal, 2021; 5(1) original article body components differences and their impact of phase angle values in athletes and non-athletes 1 vesna šeper, nebojša nešić college of applied sciences “lavoslav ružička” in vukovar, department of health studies, vukovar, croatia *corresponding author: vesna šeper, vesna.seper@vevu.hr received: jan 29, 2021; revised version accepted: mar 15 2021; published: apr 28, 2021 keywords: athletes, students, bioelectrical impedance, body mass abstract introduction: the aim of this paper was to determine pha values in athletes and sedentary population. the specific aim was to determine differences between subjects of the same sex and with a different level of physical activity, as well as the factors affecting pha values. materials and methods: sixty-six athletes and sedentary students participated in the research. they were divided into four groups according to sex and level of physical activity. routine bia at 50 khz was performed and bmi, pbf, ffm, pmm, tbw, ecw, icv, ecw/ice ratio, bmr, bm, pha and impedance were measured. results: male athletes had higher pha values (6.85±0.5°) compared to male non-athletes (6.29±0.67°), female athletes (5.61±0.44) and female non-athletes (5.47±0.58°). statistically significant differences were found in men (pha p=0.004; ecw/ice ratio p=0.002), but not in women. the highest positive correlation was found in icw (ρ+0.71 p≤0.01), while the highest negative correlation was found in impedance (ρ-0.79 p≤0.01). pha variance was mostly due to pmm (b=+0.44, p=0.002). conclusion: differences found in male athletes and non-athletes may suggest the influence of physical activity, since the variance in pha values was mostly due to pmm and a positive correlation with icw. (šeper v, nešić n. body components differences and their impact of phase angle values in athletes and non-athletes. seemedj 2021; 5(1); 89-95) seemedj 2021, vol 5, no. 1 factors impacting phase angle values 90 southeastern european medical journal, 2021; 5(1) introduction during recent years, body composition measurements have been particularly important due to the obesity epidemic and related illnesses, such as cardiovascular diseases and metabolic and endocrine disorders (1). such measurements can be useful to predict clinical outcomes in children and adults (2) and can be used to characterise physique changes during growth, aging and training. however, they sometimes require expertise regarding the measurement of skinfolds, girths and skeletal breadths (3). other methods used to asses body composition, such as computed tomography (ct), ct body composition (ctbs), magnetic resonance imaging (mri), ultrasound (us) or dual-energy x-ray absorptiometry (dexa), may not always be available or an appropriate choice (1). cost-efficient, non-invasive and simple assessment of body composition and hydration status remains an important need, which is why bioelectrical impedance (bia) is widely used (4, 5). bia is considered superior even to some serum (albumin, transferrin) or anthropometric indicators (weight change, arm muscle circumference, triceps skin-fold thickness) (6, 7). bia measures the electrical characteristics of the human body either at 50 khz or at several frequencies ranging from 1 to 1,000 khz (5). it is the only method that allows for keeping track of body hydration and cell mass using the phase angle (pha) and impedance (8). although the pha does not indicate body composition, but gives information on tissue capacity, cell size and cell membrane integrity, it correlates with cell mass, nutrition and general health both in children and adults and provides information on tissue resistance, which depends on lean body mass hydration (2). pha is obtained by measuring the ratio between impedance (r) and reactance (xc) based on the following equation (6): pha = xc/r x 180/π impedance is defined as a reduction in voltage due to the passage of current through the ionic solution of the body, while reactance is a delay in the passage of current measured as a phase shift. both measures suggest cell function (9), correlate with body shape and influence the difference in the size and function of the body (3). also, in adults, it is significant in the prognosis of the outcomes and mortality of haemodialysis, cancer, acquired immunodeficiency syndrome and cirrhosis patients (2). according to bosy-westphal et al. (10), gender and age are the main determinants of the pha in healthy adults, with men and younger people having higher pha values (10). as far as body mass is concerned, the pha increases with an increase in bmi to the value of 40 kg/m², after which it shows a negative correlation (10). in addition to age and gender, ffm and height have the strongest influence on pha values, while the ecw/icw fluid ratio shows a correlation with the pha in a clinical environment and obese people (11). body composition assessment in sports may be useful for estimating total body water (tbw) and ffm, sport performance and effects of a training program (5), whereas higher values of the pha may be linked to a higher level of physical activity (12). the potential of the pha and bioimpedance on the whole lies in the fact that it allows for non-invasive tissue monitoring, especially of hydration (1), and it can help with recommendations concerning the volume and intensity of training in sports (13). the general aim of this paper was to determine pha values in younger adult athletes and sedentary population. the specific aim was to define which body components the subjects of the same sex, but with a different level of physical activity differ in, as well as which body components relate to the pha the most. our hypothesis was that athletes will have higher pha values than non-athletes. also, we expected differences between subjects of the same sex, but with a different level of physical activity. materials and methods the research was performed at the college of applied sciences “lavoslav ružička” in vukovar in june 2017, during the competition season. a seemedj 2021, vol 5, no. 1 factors impacting phase angle values 91 southeastern european medical journal, 2021; 5(1) total of 66 student volunteers, including active male and female athletes and male and female non-athletes living a sedentary lifestyle, participated in the research. the inclusion criteria for the athletes were the following: 1) age < 35 years, 2) no medical problems according to self-reported information, 3) no smoking or alcohol abuse according to self-reported information, 4) participation in 3-5 training sessions per week and 5) involvement in organised sport activities. the inclusion criteria for the non-athletes were the following: 1) no medical problems reported 2) age < 35 years, 3) no smoking or alcohol abuse reported. subjects were divided into four groups – male athletes – ma (26 subjects), female athletes – fa (8 subjects), male non-athletes – mna (22 subjects) and female non-athletes – fna (10 subjects). the most common men’s sports were football and handball, whereas the most common women’s sports included handball and volleyball. the aim of the research and the procedure of bia measurement was explained to the students. all subjects granted their informed consent for routine bia and the research was carried out in accordance with the declaration of helsinki for medical research involving human subjects. the subjects arrived at 8:00 am for the bia exam, on an empty stomach and with an empty bladder. the measurement was performed in a standing position. the students were barefoot, wearing shorts and a t-shirt, with their hands held out and away from the body, feet slightly apart. prior to the measurement, the participants were advised to sit for 10 minutes (equilibrium period). earlier on, they had been instructed not to drink alcohol eight hours and not to consume any food four to six hours prior to the measurement. also, the subjects did not do any physical activity the day before the measurement. regarding room preparation, the examination was conducted in a lit up, pleasantly air-conditioned room (22 ºc). machine preparation included verification in accordance with the manufacturer’s instructions. the measurement was conducted on the tanita mc-780ma body mass analyser (tanita corporation, 1-14-2, maeno-cho, itabashi-ku, tokyo, japan, 2013). height was measured to the nearest 0.1 cm using a stadiometer (seca, hamburg, germany). pha and impedance were measured using bia, as well as other body components: bmi, percent body fat (pbf), ffm, tbw, extracellular water (ecw), intracellular water (icw), extracellular water/intracellular water ratio (ecw/icw ratio), percent muscle mass (pmm), bone mass (bm) and basal metabolic rate (bmr). statistical analysis results are presented as mean ± sd. normal distribution of data was evaluated using the shapiro–wilk test. differences between all four groups were determined by the kruskal–wallis h independent samples test. statistical significance was determined at the p≤0.001 level. the post hoc mann–whitney u test with bonferroni correction was done to establish the differences between male athletes and nonathletes and female athletes and non-athletes. statistical significance was set at the p≤0.03 level. the spearman’s rank correlation coefficient (ρ) was used to determine the correlation between the pha and other variables. the stepwise-backward regression was applied in order to determine which variables influenced the pha the most. statistical significance was confirmed at the p≤0.05 level. statistical analysis was performed using the ibm spss statistics 23 software (business machine corp, 2015) independently by the authors. results body composition results for all four groups are presented in table 1. the highest pha values were found in male athletes and the lowest in female non-athletes. female non-athletes had the highest pbf and male athletes the lowest pbf. the pmm and ffm were the highest in male athletes and the lowest among female nonathletes. impedance was the highest in female non-athletes, and tbw, as well as icw and ecw, were the highest in male athletes. seemedj 2021, vol 5, no. 1 factors impacting phase angle values 92 southeastern european medical journal, 2021; 5(1) table 1. values of variables for male and female athletes, male and female non-athletes and for the entire sample a g e h e ig h t b m i p b f f f m t b w e c w ic w e c w / ic w ra ti o p m m b m b m r im p e d a n c e p h a ma x̅ 20.38 184.34* 23.39* 14.49 67.82* 48.87* 18.91* 29.96* .63 64.45* 3.37* 8462.00* 558.69 6.85* nr. 26 26 26 26 26 26 26 26 26 26 26 26 26 26 sd 1.20 7.65 2.70 4.55 8.26 5.48 1.93 3.61 .03 7.87 .38 1094.69 52.35 .50 mna x̅ 20.82 181.22 23.68 16.89 63.96 46.13 18.35 27.78 .66 60.78 3.18 8003.90 586.09 6.29 nr. 22 22 22 22 22 22 22 22 22 22 22 22 22 22 sd 2.36 6.65 3.83 5.96 7.27 4.81 1.75 3.14 .03 6.93 .34 946.15 60.19 .67 p 0.97 0.08 0.80 0.16 0.16 0.14 0.46 0.052 0.002** 0.16 0.16 0.21 0.17 0.004** fa x̅ 21.63* 172.38 21.33 23.44 48.58 35.03 14.38 20.65 .69 46.11 2.46 6237.38 697.88 5.61 nr. 8 8 8 8 8 8 8 8 8 8 8 8 8 8 sd 3.93 11.12 2.36 3.67 8.46 5.96 2.69 3.29 .02 8.04 .41 981.47 97.81 .44 fna x̅ 20.00 165.70 21.59 25.32* 44.23 31.90 13.25 18.65 .71* 41.99 2.24 5781.50 750.10* 5.47 nr. 10 10 10 10 10 10 10 10 10 10 10 10 10 10 sd .82 8.25 2.15 3.73 5.98 4.21 1.90 2.36 .03 5.69 .29 698.68 94.04 .58 p 0.36 0.27 0.90 0.27 0.07 0.07 0.27 0.055 0.32 0.07 0.08 0.17 0.27 0.63 total x̅ 20.62 179.03 22.96 18.02 60.63 43.71 17.32 26.39 .66 57.60 3.02 7633.52 613.70 6.31 nr. 66 66 66 66 66 66 66 66 66 66 66 66 66 66 sd 2.08 10.26 3.11 6.33 11.82 8.29 2.95 5.39 .04 11.26 .59 1417.75 98.39 .77 p 0.74 0.000† 0.09 0.000† 0.000† 0.000† 0.000† 0.000† 0.000† 0.000† 0.000† 0.000† 0.000† 0.000† *the highest values; **statistical significance p≤0.03 †statistical significance p≤0.001 (ma – male athletes; fa – female athletes; mna – male non-athletes; fna – female non-athletes; x ̅ – arithmetic mean; nr. – number; sd – standard deviation; p – statistical significance; bmi – body mass index; pbf – percent body fat; ffm – fat free mass; tbw – total body water; ecw – extracellular water; icw – intracellular water, ecw/icw ratio – extracellular water/intracellular water ratio; pmm – percent muscle mass; bm – bone mass; bmr – basal metabolic rate; pha – phase angle seemedj 2021, vol 5, no. 1 factors impacting phase angle values 93 southeastern european medical journal, 2021; 5(1) ecw/icw ratio was the highest among female non-athletes and the lowest among male athletes. the amount of bm was the highest in male athletes and the lowest in female nonathletes. male athletes had the highest bmr. statistically significant differences between the male subjects existed only in the ecw/icw ratio (p=0.002) and pha values (p=0.004). as far as the female subjects are concerned, whether athletes or those leading a sedentary lifestyle, there were no statistically significant differences (p˃0.03). regarding the correlation between the pha and other variables, there was a positive correlation with height (ρ+0.35 p=0.004), bmi (ρ +0.55 p≤0.01), ffm (ρ+0.64 p≤0.01), pmm (ρ+0.64 p≤0.01), tbw (ρ+0.66 p≤0.01), ecw (ρ+0.58 p≤0.01) and icw (ρ+0.71 p≤0.01), bm (ρ+0.63 p≤0.01) and bmr (ρ+0.64 p≤0.01) and a negative correlation with pbf (ρ-0.35 p=0.004), ecw/icw ratio (ρ-0.78 p≤0.01) and impedance (ρ-0.79 p≤0.01). regression analysis showed that pha values were mostly influenced by pmm (b=+0.44, p=0.002), bmi (b=+0.31, p=0.000) and impedance (b=+0.004, p=0.03), which describes almost 83% of the variance in the values of the pha (r²=0.83). discussion pha is a good prognostic indicator in numerous clinical conditions of patients suffering from hiv, bacterial infection, liver cirrhosis, kidney disease, tuberculosis and cancer, but little is known about pha values in healthy individuals (6). according to selberg and selberg (8), pha values over 5.4° are considered normal, in the range of 4.4° to 5.4° as borderline and under 4.4° as abnormal (8). lower values usually indicate a cell integrity disorder or even cell death (14). in this research, pha values of male athletes were 6.85±0.5°, female athletes 5.61±0.44°, male nonathletes 6.29±0.67° and female non-athletes 5.47±0.58°. a potential explanation may be that higher pha values are present in physically active people (15), but in the absence of sportspecific reference values, we can only use general healthy population values as references and hypothesise on the influence of physical activity (16). in their systematic review on bioelectrical impedance phase angle in sports, di vincenzo et al. (5) stated that pha values have been shown to be significantly associated with muscle strength and physical activity and to vary between sexes and with age (5). since the pha is considered a simple indicator of muscle mass and is defined by tissue hydration and cell membrane potential (8), and since ecw/icw ratio is one of the measures that influences pha variability, changes in pha values usually correlate with cell size, cell permeability and differences in fluid distribution in various tissue types (11). lower pha values are caused by an increase in the ecw/icw ratio in patients with inflammatory conditions and obese individuals (11). male athletes in our research had the lowest values of the ecw/icw ratio and pbf among all four groups and the highest values of ffm and pmm. we also found statistically significant differences in pha values and the ecw/icw ratio between male athletes and non-athletes. however, no such differences were found among the female subjects, which may be due to pbf values. according to gallagher et al. (17), pbf of healthy women aged 20-39 ranges from 21% to 33% and of athletes from 14% to 20% (18). on the other hand, female athletes in this research had pbf values of 23.44±3.67%, while female non-athletes had pbf values of 25.32±3.73%, with a mean difference of 1.88±0.06%, placing them in the same reference category. we found a strong positive correlation between the pha and icw, tbw, ffm, pmm, bmr, bm, ecw and bmi, as well as a strong negative correlation with impedance and the ecw/icw ratio. differences in the distribution of fluids, an increase in the amount of ecw and a compensatory increase in the ecw/icw ratio may be leading to a decrease in pha values (11). increase in the ecw/icw ratio leading to a decrease in pha values may be connected to pbf because adipose tissue influences haemodynamics or fluids (11), although in our research, pbf showed a moderate correlation to the pha. higher icw content in physically active people may be a reflection of physiological cellular adaptations leading to higher pha values (12). genton et al. (12) hypothesised that physically active people practice carbohydrate loading to improve performance and seemedj 2021, vol 5, no. 1 factors impacting phase angle values 94 southeastern european medical journal, 2021; 5(1) consequently have higher icw content to store glycogen (12). silva et al. (19) state that regardless of body composition changes, athletes who increase reactance and resistance reduce ecw, while those who raise pha increase icw (19). the correlation with ffm and pmm may be explained by the fact that the pha is directly related to the amount and function of cell membranes, in this case, muscle cells (6). as for bmi, it explained 31% of the variance in pha values and according to bosy-westphal (10), an increase in pha values with an increase in bmi only occurs with a bmi of about 40 kg/m² and is just a reflection of an increased number of muscle or fat cells (10). bmr also showed a positive correlation with the pha, which may be linked to greater amounts of ffm in athletes (20). regarding bm, we found no evidence explaining a moderate positive correlation with the pha, except that it may be due to the age of the participants. negative correlation of the pha with impedance may be due to sex. men tend to have lower values of impedance than women (15) due to a higher amount of ffm and lower pbf. our results are only applicable when using a bia device operating at a single frequency (50 khz), which can be a limiting factor. for a better estimate of the influence of body components on the pha, the bia measurement should be performed in different frequency ranges (5 khz, 50 khz, 100 khz) (15). another limiting factor is that we had no record of the nutritional status of the subjects. moreover, regarding the medical condition of the students, we relied on selfreported information. a small sample size may also be a limitation, even though the student athletes played different sports. in conclusion, pha values in this research were higher in athletes than in non-athletes and higher in male athletes compared to female athletes. we found significant differences in pha values and the ecw/icw ratio between male athletes and non-athletes, but no such differences were found in the female subjects. variables showing the highest positive correlation with the pha were icw, tbw, ffm and pmm, indicating a possible influence of physical activity. regression analysis in this research showed that the variance in the pha is mostly influenced by pmm, bmi and impedance. acknowledgement. results of this research were presented at the 14th international scientific conference of sport kinetics titled “movement in human life and health” in poreč, croatia, in june 2018. the authors would like to thank the volunteers for participating in this research. special thanks to tomislava vijant, teacher of english and ma (master of arts) in translation studies for her help with the translation.. disclosure funding. no specific funding was received for this study. transparency declaration: competing interests: none to declare references 1. andreoli a, garaci f, cafarelli fp, guglielmi, g. body composition in clinical practice. eur j radiol. 2016; 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(2020). usefulness of raw bioelectrical impedance parameters in tracking fluid shifts in judo athletes. eur j sport sci. 2020; 20(6):734-743. https://doi.org/10.1080/17461391.2019.1668481 20. jagim ar, camic cl, kisiolek j, luedke j, erickson j, jones mt, oliver jm. (2018). accuracy of resting metabolic rate prediction equations in athletes. j strength cond res. 2018; 32(7):1875-1881. doi: 10.1519/jsc.0000000000002111 1 author contribution. acquisition of data: šeper v, nešić n administrative, technical or logistic support: šeper v, nešić n analysis and interpretation of data: šeper v, nešić n conception and design: šeper v, nešić n critical revision of the article for important intellectual content: šeper v, nešić n drafting of the article: šeper v, nešić n final approval of the article: šeper v, nešić n guarantor of the study: šeper v, nešić n, provision of study materials or patients: šeper v, nešić n statistical expertise (statistical analysis of data): šeper v, nešić n seemedj 2022, vol 6, no 1 iodine intake 12 southeastern european medical journal, 2022; 6(1) review article overview of iodine intake 1 vanja vasiljev 1*, alen subotić 2, mihaela marinović glavić 1, denis juraga 1, lovorka bilajac 1,3, bojan jelaković 4,5, tomislav rukavina 1,3 1 university of rijeka, faculty of medicine, department of social medicine and epidemiology, rijeka, croatia 2 institute of emergency medicine of zagreb county, zagreb, croatia 3 teaching institute of public health of primorje-gorski kotar county, rijeka, croatia 4 university hospital centre zagreb, department of nephrology, arterial hypertension, dialysis and transplantation, zagreb, croatia 5 university of zagreb, school of medicine, zagreb, croatia *corresponding author: vanja vasiljev, vanjav@uniri.hr received: mar 1, 2022; revised version accepted: apr 7, 2022; published: apr 27, 2022 keywords: iodine, sodium chloride, dietary, diet abstract iodine is an essential element for human health. food is the primary source of iodine, but the iodine content of local foods depends on the iodine content of the soil. therefore, a low iodine concentration in soil and water results in plants and animals with low iodine content. numerous effects of iodine deficiency on growth and development are known as iodine deficiency disorders. iodine deficiency has been identified as the most common cause of brain damage in the world and is linked to its effects on infant and child growth and development. supplementation of table salt with iodine was introduced in the 20th century. croatia was one of the first countries to introduce the supplementation of table salt with potassium iodide at a concentration of 10 mg/kg in 1953 and 25 mg/kg in 1993. in 2003, the croatian population reached iodine sufficiency, but given the excessive salt intake (11.6 g/day) and additional sources of iodine in the diet, the question arises, are we consuming too much iodine? this article gives a short overview of iodine intake. (vasiljev v, subotić a, marinović glavić m, juraga d, bilajac l, jelaković b, rukavina t. overview of iodine intake. seemedj 2022; 6(1); 12-20) seemedj 2022, vol 6, no 1 iodine intake 13 southeastern european medical journal, 2022; 6(1) discovery of iodine and history of iodine supplementation purple vapour was first discovered in 1811 by the chemist courtois during the production of saltpeter. the production of saltpeter required soda, which was obtained from the ashes of seaweed. the chemical reaction required to make saltpeter resulted in the formation of insoluble material at the bottom of metal vats. the material was cleaned out using acid and heat, which led to purple vapour crystallising on the bottom of the vats. however, iodine was only discovered two years later by sir humphry davy and gay-lussac (1). the use of iodine in medicine started in 1819 with coindet, a physician from geneva who administered a tincture of iodine to goitre patients, which resulted in the swelling going down within a week. the link between iodine and the environment was not confirmed, although the french chemist chatin proved that the iodine content in water and food was insufficient in the areas commonly affected by goitre. the first paper on the link between iodine deficiency and goitre was published in 1851 (2). chatin’s discoveries on the link between environment and iodine deficiency were neglected until the end of the 19th century. in 1896, the presence of iodine in the thyroid gland was discovered by baumann and roos (3). switzerland was the first country to introduce iodine as prophylaxis against goitre and cretinism. since 1922, switzerland has had a continuous program of supplementation of salt with iodine, with the level of iodine eventually being raised from 15 mg/kg in 1980 to 20 mg/kg in 1998 (3, 4). role of iodine, iodine deficiency disorders and optimal iodine intake the thyroid gland of a healthy adult stores 7080% of the total iodine content in the body, which ranges from 15 to 20 mg. it also uses about 80 µg of iodine per day for the synthesis of thyroid hormones. iodine is essential for the synthesis and production of thyroid hormones (triiodothyronine – t3 and thyroxine – t4) and for normal thyroid function. the thyroid hormones control the metabolic processes in our body. their production is controlled and influenced by the pituitary gland and its hormone thyrotropin (thyroid-stimulating hormone – tsh). tsh increases the uptake of iodine from the blood into the thyroid gland and stimulates the production of thyroid hormones, which is regulated through feedback. when the level of thyroid hormones in the blood is low or decreases, increased tsh is released from the pituitary gland. tsh stimulates the function of the thyroid gland, causes cell growth and proliferation of thyroid tissue, and, in case of chronic iodine deficiency, can lead to an enlargement of the thyroid gland known as a goitre. goitre is one of the most common dietrelated diseases (5). in addition, when iodine intake is very low, thyroid hormone production decreases despite elevated tsh levels, leading to hypothyroidism (6, 7). numerous effects of iodine deficiency on growth and development are known as iodine deficiency disorders. iodine deficiency has been identified as the most common cause of brain damage in the world and it is related to its impact on the growth and development of infants and children (8,9). table 1. recommended daily intake of iodine (12) children 0-5 years children 6-12 years adults pregnant and lactating women intake (μg) 90 120 150 250 the spectrum of iodine deficiency disorders includes mental retardation, hypothyroidism, goitre and varying degrees of other growth and developmental disorders (10). chronic iodine deficiency is also associated with an increased risk of developing follicular thyroid cancer (11). seemedj 2022, vol 6, no 1 iodine intake 14 southeastern european medical journal, 2022; 6(1) according to zimmerman and andersson, the recommended daily dietary intake for iodine is as shown in table 1. iodised salt regulations one of the greatest public health challenges worldwide is the lack of essential vitamins and minerals in the daily diet. this issue is widespread among women and young children in lowand middle-income countries (13). this can lead to serious health issues and economic consequences, which only adds to the global burden of disease (14,15). this also applies to iodine and the issue of high prevalence of disorders caused by insufficient iodine intake (16). to overcome this issue, many national nutrition strategies started promoting programs to eliminate iodine deficiency disorders in the 1990s, after the world summit for children and the joint united nations children’s fund (unicef)/who committee on health recognised the benefits of iodisation of table salt (17-19). iodisation of salt has been recognised as the best preventive measure to eliminate iodine deficiency disorders at the population level (20). not only is the iodisation of salt technically feasible, but salt is also consumed in standard quantities by all segments of the population worldwide (21). between 1942 and 2021, 123 countries worldwide established a legal framework for mandatory iodisation of table salt and 21 countries introduced legislation for voluntary iodisation of table salt (22). according to estimates made by unicef, an average of 88.7% of households worldwide consumed table salt with some form of iodine in 2021. most households using iodised table salt are in the east asia and pacific region (92%) and in south asia (89.9%) (23). on the other hand, there are also countries with excessive iodine intake, such as south korea (449 µg/l), djibouti (335 µg/l), cameroon (>300 µg/l), honduras (356 µg/l) and colombia (407 µg/l). the reasons for the increased iodine concentration are related to diet, groundwater and drinking water, which are naturally rich in iodine, as well as to high amounts of iodine added to salt considering the per capita intake of salt (22). although the legal regulations on compulsory or voluntary iodine intake via table salt have greatly reduced the issue of iodine deficiency disorders, there are still regions where almost one billion people do not have access to iodised table salt(22). one way to ensure the necessary supply of iodised table salt for such persons is to promote the universal salt iodization (usi) initiative, which is one of the most economical, convenient and effective strategies to increase the intake of iodised table salt (24). the initiative could be improved through cooperation between relevant stakeholders at local, regional and national levels, in the food industry and in the scientific community. nowadays, eu member states introduce various strategies and legal frameworks to combat iodine deficiency, but there are still countries such as norway (75 µg/l), finland (96 µg/l) and germany (89 µg/l) that are deficient in iodine from table salt (22). to combat iodine deficiency, it is necessary to identify potential barriers and adopt a uniform approach to the development of a single regulatory framework at the eu level based on the guidelines of who, without exception. furthermore, such a uniform approach should allow national governments to implement measures in line with their culture and to remove barriers to marketing for the purpose of raising awareness of iodine deficiency disorders among the general public (25). 1. dietary sources of iodine and fortification of salt with iodine unlike most essential nutrients, the status of iodine in our body is not related to socioeconomic factors, but rather to the climate in which we live(16). the iodine content of local foods depends on the iodine content in the soil. therefore, a low concentration of iodine in the soil and water will result in plants and animals with low iodine content. most of the iodine on our planet is found in the oceans. also, the iodine seemedj 2022, vol 6, no 1 iodine intake 15 southeastern european medical journal, 2022; 6(1) content in the soil varies depending on the region. if the soil is older and more exposed to external factors, it is more likely that iodine will be washed out by soil erosion. people who depend on local foods produced in iodinedeficient areas must also rely on foods fortified with iodine(26, 27). humans receive iodine through food, food supplements and water, mainly in the form of iodide (27, 28). iodine concentrations vary both between food groups and within the groups themselves. foods containing iodine include seafood, eggs, milk and dairy as well as iodised salt. the iodine content of milk and eggs depends on how animals are fed (iodine-enriched feed) and on the hygiene on the farm (28). the natural iodine content in most foods and beverages is low, and the most commonly consumed foods provide 3 to 80 µg per meal (5). in croatia, all table salt used for food is iodised by adding 25 mg of iodine per kilogram of salt in order to prevent diseases related to insufficient iodine intake and croatia is recognised as an iodine sufficient country (29, 30). the urinary iodine test is a wellknown, inexpensive and easily accessible method of determining iodine status (31). a nationwide project named epidemiology of hypertension and salt intake (eh-uh 2) is currently active in croatia. one of its main objectives is to determine iodine intake from a 24-hour urine sample and to assess whether there is a risk of exposure to low iodine concentrations if the recommended daily salt intake limit of 5 grams is observed (strategic plan for reduction of salt intake) (32, 33). iodine toxicity excessive iodine intake usually occurs when people take iodine supplements to improve thyroid function. several types of seafood, including shrimp, cod, tuna and seaweed, are rich in iodine. in cultures where a lot of seaweed is eaten, people sometimes consume thousands of milligrams of iodine per day. it is estimated that people in japan consume between 1,000 and 3,000 mg of iodine daily, mainly from seaweed. this leads to iodine-induced hyperthyroidism and goitre in japan. however, the same research also points out that the higher iodine intake may play a role in the low cancer rates and high life expectancy in japan (8). exceeding the maximum permissible amount of iodine can lead to poisoning and iodine toxicity may eventually lead to iodine goitre, hypothyroidism, or myxoedema (35). also, excessive iodine intake can have a negative impact on patients with breast cancer due to the stimulation of the transcriptional activity of oestrogen receptor α (er-α), resulting in an elevated risk of developing thyroid cancer (36). chronic toxicity develops only if iodine intake exceeds 2 mg/day. in some sensitive individuals, ingestion of iodine-containing substances may lead to thyroid dysfunction due to high iodine exposure. under certain circumstances, excessive iodine intake can have harmful effects on the thyroid gland after just one exposure to an iodine-containing substance (35). patients with iodine deficiency and patients with a pre-existing thyroid condition may be sensitive to iodine levels considered safe for the general population. neonates, older people and pregnant women may also be more susceptible to iodine excess (37). it is very difficult to get iodine poisoning from food alone, so iodine poisoning is usually a result of taking too many iodine supplements (38). acute iodine poisoning is rare and the symptoms of iodine poisoning range from relatively mild to severe, depending on the amount of iodine. mild symptoms of iodine poisoning include diarrhoea, burning in the mouth, nausea and vomiting. very large amounts of iodine can cause a metallic taste in the mouth, increased salivation, irritation of the digestive system and acne-like skin changes. severe symptoms of iodine poisoning include swelling of the airways, blue skin discoloration (cyanosis) and low heart rate. iodine poisoning can also lead to kidney failure (39). certain medications can also increase the amount of iodine in the body. amiodarone, a drug used to regulate heart rate and rhythm, contains 75 mg of iodine in each 200 mg tablet, which is one hundred times more than the standard recommended daily intake (40). various drugs, such as propranolol (in high seemedj 2022, vol 6, no 1 iodine intake 16 southeastern european medical journal, 2022; 6(1) doses), the anti-thyroid drug propylthiouracil, dexamethasone, the cholecystographic agents (ipodate and iopanoic acid) and the previously mentioned amiodarone can inhibit the conversion of t4 into t3 (41). potassium iodide supplements and the contrast medium used for ct scans also contain iodine (9). elemental iodine is an oxidising irritant and can cause lesions in case of direct contact with the skin, while exposure to iodine vapours causes irritation of the lungs, eyes and skin. diagnosis and assessment of iodine toxicity is an important part of the health care team’s approach to providing treatment, enhancing care coordination and establishing communication necessary to improve patient outcomes. iodine toxicity is a rare condition that requires a comprehensive initial diagnosis and a heightened level of suspicion. patients may present with vague signs and symptoms. although medical history may reveal toxicity, the cause is difficult to determine without further investigation. the consequences of iodine toxicity depend on the cause and severity. however, to improve outcomes, it is recommended that an interprofessional group of experts be consulted to monitor the patient’s vital signs and educate the patient and their family (42). discussion iodine is an essential mineral for healthy functioning of the human body, but only in moderate amounts. iodine boosts thyroid function and consequently increases metabolism, supports a healthy pregnancy and prevents cretinism, and promotes heart health by stimulating the production of hormones that regulate heart rate and blood pressure. over one billion people around the world still do not have access to iodised salt and therefore suffer from iodine deficiency (10, 11). after the second world war, the prevalence of goitre and cretinism in the endemic areas of the republic of croatia was high (43, 44). the highest prevalence was found in the village of rude and in the samobor and žumberak mountains, where almost 85% of school children suffered from goitre and 2.3% of children suffered from cretinism. the first intervention involving iodised salt was made in 1953 and set at 10 mg per kilogram of salt. this intervention led to a threefold reduction in goitre in children and complete elimination of cretinism (44, 45). fifteen kilometres from the adriatic coast, in the region of grobnik, croatia, goitre was endemic before iodised salt prophylaxis. in 1963, the prevalence of goitre was 63% in school children and 34% in adults. a second survey was conducted in 1981 and the prevalence of goitre was 18% in school children and 11% in adults. in 2001, another survey on the prevalence of goitre was conducted in the grobnik region. the results showed that the prevalence of goitre was 6.6% among school children and 6.4% among adults. a significant decrease in prevalence was achieved among school children, but not among adults due to the hereditary thyroid disease in the indigenous population (11.7%) (46). the high prevalence of goitre led to an increase in the potassium iodide content in salt from 10 mg/kg to 25 mg/kg (47). in the period from 2002 to 2009, urine samples of school children were analysed to determine the iodine concentration using a median urinary iodine concentration [uic] of 68 μg/l. the results showed that the iodine concentration in 2009 was significantly higher than in 2002, indicating that there were hidden sources of iodine in the diet besides salt (29). the first legal document regulating the general iodisation of salt in the republic of croatia was introduced in 1953, prescribing 10 mg of potassium iodide (ki) per kilogram of salt (48). in the 1990s, at the initiative of the then chairman of the national committee for eradication of goitre and control of iodine prophylaxis, professor zvonko kusić, two new legal documents were introduced: instructions on iodisation of table salt (official gazette 84/96) and the salt iodisation regulation (official gazette 15/97), which led to a further increase in the prescribed amount of iodine in table salt (48). the initiative for universal iodisation of salt in croatia allows for iodisation of table salt to be applied at three levels: in households, food industry and animal feed production. the croatian model of table salt iodisation complies seemedj 2022, vol 6, no 1 iodine intake 17 southeastern european medical journal, 2022; 6(1) with all guidelines of the who, international council for the control of iodine deficiency disorders (iccidd) and unicef and it has been internationally recognised as one of the models for addressing this emerging public health issue (48, 49). conclusion this article provides an overview of iodine discovery and its physiological role in our bodies, at the same time explaining the optimal iodine intake in relation to different dietary sources and environments. iodine toxicity and iodine deficiency disorders are also addressed, which raises the question of whether we really know how much iodine we consume per day. another question is, if we ingest too much salt, are we placing ourselves at the risk of excessive iodine intake? the answer to those questions could be found in different initiatives and projects, such as the eh-uh 2, for which a comprehensive and detailed strategic approach should be defined to assess the amount of iodine intake across the population. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. kelly fc. iodine in medicine and pharmacy since its discovery-1811-1961. proc r soc med. 1961;54(10):831-6. 2. chatin a. recherches sur l'iode des eaux douces;de la presence de ce corps dans les plantes at les animaux terrestes. c r acad sci paris. 1851.;31:280-3. 3. zimmermann mb. research on iodine deficiency and goiter in the 19th and early 20th centuries. j nutr. 2008;138(11):2060-3. doi: 10.1093/jn/138.11.2060. 4. bürgi h. the swiss legislation on iodized salt. idd newsletter. 1999;15:57-8. 5. delange f. the disorders induced by iodine deficiency. thyroid. 1994; 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104(3):868s-76s. doi: 10.3945/ajcn.115.110064. 29. flachowsky g, franke k, meyer u, leiterer m, schone f. influencing factors on iodine content of cow milk. eur j nutr. 2014;53(2):351-65. doi: 10.1007/s00394-013-0597-4. 30. kusić z, jukić t, rogan a, juresa v, dabelic n, stanicic j, et al. current status of iodine intake in croatia the results of 2009 survey. coll antropol. 2012; 36:123-8. seemedj 2022, vol 6, no 1 iodine intake 19 southeastern european medical journal, 2022; 6(1) 31. kusić z, novosel sa, dabelić n, punda m, roncević s, labar z, lukinac lj, nöthig-hus d, stanicić a, kaić-rak a, mesaros-kanjski e, karner i, smoje j, milanović n, katalenić m, juresa v, sarnavka v. croatia has reached iodine sufficiency. j endocrinol invest. 2003; 8:738-42. 32. who. urinary iodine concentrations for determining iodine status in populations geneva: world health organization 2013 [cited 11.01.2022.]. available from: http://www.who.int/nutrition/vmnis/indicators /urinaryiodine. 33. jelaković b. epidemiologija hipertenzije u hrvatskoj. croatia: hrzz. available from: https://hdh.emed.hr/projekti/eh-uh. 34. strateški plan za smanjenje prekomjernog unosa kuhinjske soli u republici hrvatskoj 2015.2019. hrvatski zavod za javno zdravstvo; 2015. 35. zava tt, zava dt. assessment of japanese iodine intake based on seaweed consumption in japan: a literature-based analysis. thyroid res. 2011; 4:14. doi: 10.1186/1756-6614-4-14. 36. koukkou eg, roupas nd, markou kb. effect of excess iodine intake on thyroid on human health. minerva med. 2017;108(2):136-46. doi: 10.23736/s0026-4806.17.04923-0. 37. he s, wang b, lu x, miao s, yang f, zava t et al. iodine stimulates estrogen receptor singling and its systemic level is increased in surgical patients due to topical absorption. oncotarget. 2017;4;9(1):375-384. doi: 10.18632/oncotarget.20633 38. ershow ag, goodman g, coates pm, swanson ca. assessing iodine intake, iodine status, and the effects of maternal iodine supplementation: introduction to articles arising from 3 workshops held by the nih office of dietary supplements. am j clin nutr. 2016;104 suppl 3:859s-63s. doi: 10.3945/ajcn.115.111161. 39. nettore ic, colao a, macchia pe. nutritional and environmental factors in thyroid carcinogenesis. int j environ res public health. 2018;15(8):1735. doi: 10.3390/ijerph15081735. 40. bulloch mn. acute iodine toxicity from a suspected oral methamphetamine ingestion. clin med insights case rep. 2014; 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[updated 2022 jan 9]. in: statpearls [internet]. treasure island (fl): statpearls publishing; 2022 jan-. available from: https://www.ncbi.nlm.nih.gov/books/nbk560 770/. 44. kusić z, lelas v, drenjančević-perić i, antolić b, katalenić m, gross-bošković a. 2009. znanstveno mišljenje o važnosti konzumiranja jodirane soli u rh. hrvatska agencija za hranu. 45. kusić z, jukić t. history of endemic goiter in croatia: from severe iodine deficiency to iodine sufficiency. coll antropol. 2005; 29(1):9-16. 46. jukić t, dabelić n, rogan sa, nõthig-hus d, lukinac lj, ljubicić m, kusić z. the story of the croatian village of rude after fifty years of compulsory salt iodination in croatia. coll antropol. 2008;32:1251-4. 47. crncević-orlić z, ružić a, rajković-molek k, kapović m. the effectiveness of a 40-year long iodine prophylaxis in endemic goitre region of grobnik, croatia. coll antropol. 2005; 29:509-13. 48. vucinic m, kusec v, dundovic s, ille j, dumic m. the effect of 17 years of increased salt iodization on the prevalence and nature of goiter in croatian schoolchildren. j pediatr endocrinol metab. 2018;31(9):995-1000. doi: 10.1515/jpem2018-0129. 49. kusić z, katalenić m, drenjančević i, jelaković b. znanstveno mišljenje o mogućnosti jodiranja pustinjske soli. zagreb: hrvatska agencija za hranu; 2016. seemedj 2022, vol 6, no 1 iodine intake 20 southeastern european medical journal, 2022; 6(1) 1 author contribution. acquisition of data: vasiljev v, subotić a, marinović glavić m, juraga d, bilajac l, jelaković b, rukavina t administrative, technical or logistic support: vasiljev v, subotić a, marinović glavić m, juraga d, bilajac l, jelaković b, rukavina t analysis and interpretation of data: vasiljev v, subotić a, marinović glavić m, juraga d, bilajac l, jelaković b, rukavina t conception and design: vasiljev v, subotić a, marinović glavić m, juraga d, bilajac l, jelaković b, rukavina t critical revision of the article for important intellectual content: vasiljev v, subotić a, marinović glavić m, juraga d, bilajac l, jelaković b, rukavina t drafting of the article: vasiljev v, subotić a, marinović glavić m, juraga d, bilajac l, jelaković b, rukavina t final approval of the article: vasiljev v, subotić a, marinović glavić m, juraga d, bilajac l, jelaković b, rukavina t provision of study materials or patients: vasiljev v, subotić a, marinović glavić m, juraga d, bilajac l, jelaković b seemedj 2022, vol 6, no 1 arterial hypertension, risk of mortality and covid-19 74 southeastern european medical journal, 2022; 6(1) review article arterial hypertension and risk of mortality in patients with covid-19 infection 1 nikolina bukal 1, melanija kolarić 2, ines golubić 3, josipa josipović 4,5, vedran premužić 6, ana jelaković 6, sandra karanović 5,6*, nikolina bašić jukić 5,6, bojan jelaković 5,6 1 department of internal medicine, nephrology and pulmonology, general hospital ”dr. josip benčević”, slavonski brod 2 department of nephrology and dialysis, general hospital varaždin 3 department of nephrology and endocrinology, general hospital “dr. tomislav bardek”, koprivnica 4 department of nephrology and dialysis, university hospital centre sisters of mercy, zagreb 5 school of medicine, university of zagreb 6 department of nephrology, arterial hypertension, dialysis and transplantation, university hospital centre zagreb *corresponding author: sandra karanović, skaranov@kbc-zagreb.hr received: feb 27, 2022; revised version accepted: apr 23, 2022; published: apr 27, 2022 keywords: arterial hypertension; covid-19 infection; renin–angiotensin system abstract covid-19 is currently a major global health concern. among many unanswered questions related to covid-19, some of the most debated ones are those concerning arterial hypertension. arterial hypertension is a major risk factor for mortality worldwide and its importance has been emphasised even further in light of covid-19. the most common antihypertensive drugs are ace inhibitors and angiotensin ii type-i receptor blockers. sars-cov-2 utilises the angiotensin-converting enzyme-2 (ace2) for cell entry and therefore has a direct effect on the renin–angiotensin system (ras). in terms of arterial hypertension and covid-19, there are three main issues which have been the focus of extensive debates. first, is arterial hypertension a predisposing factor for covid-19 infection? second, does arterial hypertension affect the severity of covid-19 infection and increase the risk of all-cause and cardiovascular mortality? and finally, how important is the interaction of covid-19 infection and the renin–angiotensin system for clinical outcomes? is ras blockade beneficial or harmful? the aim of this brief review was to provide substantiated answers to these questions. (bukal n, kolarić m, golubić i, josipović j, premužić v, jelaković a, karanović s*, bašić jukić n, jelaković b. arterial hypertension and risk of mortality in patients with covid-19 infection. seemedj 2022; 6(1); 74-82) seemedj 2022, vol 6, no 1 arterial hypertension, risk of mortality and covid-19 75 southeastern european medical journal, 2022; 6(1) introduction infection with sars-cov-2 is associated with high mortality rates and it is currently the most important global health concern. the risk of severe outcomes of covid-19 infection increases with patients’ age and prevalence of comorbidities (1). arterial hypertension is one of the most prevalent chronic conditions (2) and it is often treated with renin–angiotensin system (ras) blockage. angiotensin-convertingenzyme 2 (ace2) is the pivotal receptor for sars-cov-2 to enter host cells (3) and as such, it provides a link between covid-19 and ras. the association between arterial hypertension, its treatment, the risk of sars-cov-2 infection and clinical outcomes of covid-19 infection has been a concern and a matter of debate for both patients and physicians. the aim of this paper was to review our knowledge about this issue by focusing on the most important publications issued so far and by answering the following key questions. first, does arterial hypertension predispose individuals to covid-19 infection? second, does arterial hypertension increase the risk of all-cause and cardiovascular mortality? third, how important is the interaction of covid19 infection with the renin–angiotensin system? and finally, is ras blockade beneficial or harmful in respect to covid-19 outcomes? however, before discussing arterial hypertension and covid-19 infection, it is necessary to point out some important facts about arterial hypertension, ras and respiratory infections independent of covid-19 infection. it is well known that people over the age of 65 have a higher risk of a lower respiratory tract infection (lrti) as well as a higher prevalence of arterial hypertension (4,2). in addition, a beneficial effect of ras blockade associated with lrti was detected in terms of lower incidence and lower mortality rates (5). based on the above, it can be concluded that arterial hypertension is not an independent risk factor for lrti and that the use of ras blockers decreases the incidence and improves the outcomes of lrti (6). is arterial hypertension a predisposing factor for covid-19 infection? according to the first data collected in lombardy in early 2020, almost 73% of patients with covid-19 had arterial hypertension (7). at that point, arterial hypertension was often indicated as a risk factor for covid-19, together with cardiovascular and cerebrovascular diseases, chronic kidney disease, malignancies and obesity. however, it was initially overlooked that the average age in the lombardy group was 81. having that in mind, it is not surprising that the prevalence of arterial hypertension was so high. other data from the first reports from lombardy were related to the intake of antihypertensive drugs. almost 47% of covid-19 patients were taking ras blockers. this information, along with some pathophysiological considerations, aroused suspicion that drugs which block ras could be related to the higher incidence of sars-cov-2 infection, more severe forms of the disease and increased mortality. to support those data, mancia et al. analysed the data concerning 6,272 people with covid-19, together with a control group of 30,759 subjects paired based on age and gender (8). the first important fact they discovered was that the covid-19 patients more often had cardiovascular diseases in their medical history, especially coronary heart disease and heart failure. moreover, they also suffered from respiratory diseases, chronic kidney disease and, to some extent, carcinomas. the second important piece of data concerned the association between ras blockers and covid-19 infection – no significant correlation of ras blockers with covid-19 infection was detected in the study, neither with regard to the severity of the clinical features of covid-19 infection nor with regard to age or gender. in relation to covid-19 in this group of subjects, ras blockers had the same status as all other seemedj 2022, vol 6, no 1 arterial hypertension, risk of mortality and covid-19 76 southeastern european medical journal, 2022; 6(1) classes of antihypertensive drugs. the final conclusion was that the observed relationship between arterial hypertension and covid-19 infection was blurred by the influence of age and comorbidity. a number of other studies have confirmed that the prevalence of arterial hypertension in covid-19 patients does not differ significantly from the prevalence in the general population (9). does arterial hypertension affect the severity of covid-19 infection and increase mortality? a number of researchers have noticed that patients with more severe forms of covid-19 infection suffer from arterial hypertension significantly more frequently (table 1). by analysing a group of patients from wuhan, china, shi et al. found that covid-19 mortality was significantly higher in patients with myocardial damage and that arterial hypertension was also much more common among them (10). in a retrospective study by wu et al., patients with hypertension were more likely to have increased oxygen demand, myocardial injury and a greater risk of developing severe covid-19, suggesting that hypertension might play an important role in covid-19 (11). these findings are consistent with the idea that hypertension cases involve an increased risk of comorbidity, infection and multiple organ function damage (12,13). in a multicentre retrospective cohort study of 1,833 covid-19 patients, mubarik et al. found that the prevalence of hypertension was 40.5% and that patients with hypertension were more likely to have severe covid-19 illness than patients without hypertension (14). evidence from a meta-analysis which included 24 observational studies with 99,918 covid-19 patients suggested that hypertension was independently associated with a significantly increased risk of critical covid-19 and in-hospital mortality of covid-19 (15). guan et al. detected a higher risk of severe covid-19 in older individuals and those with underlying health conditions such as arterial hypertension (16). gao et al. observed that mortality was much higher in patients with uncontrolled hypertension compared to those who achieved target blood pressure values (17). they also noticed that mortality was significantly lower in patients treated with ras blockers. interesting results were obtained from the hope covid-19 registry (italy, spain, germany), where out of 5,937 patients with covid-19 infection, 48.8% had arterial hypertension and 70.3% were treated with ras blockers (18). as in the lombardy group, the patients with covid-19 infection more frequently had comorbidities associated with an increased risk of death. over 40 days of follow-up, a significantly higher mortality rate was noticed among patients with arterial hypertension (29.6% vs 11.3%; p<0.001). based on this finding, the authors expected that the increased mortality in covid-19 infection could be related to arterial hypertension. however, when data was analysed in more detail using the multivariate cox regression analysis, the authors found that the age over 65, respiratory infections and sepsis were independent predictors of mortality. a very important finding derived from their analysis was the fact that the use of ras blockers had a protective effect on mortality, consistent with the findings by gao et al. (17). sheppari et al. have recently published an interesting finding concerning a group of patients with stage 1 uncontrolled arterial hypertension that there were no differences in mortality compared to the patients with controlled hypertension (19). they did not observe any associations between the control of arterial hypertension and covid19 infection or the need for hospitalisation. they concluded that the association between arterial hypertension control and mortality could be explained by advanced atherosclerosis and preexisting target organ damage. the authors pointed out a probable key problem associated with arterial hypertension and covid-19 infection, and that is the issue of irregular medical check-ups. additionally, it should not be forgotten that the immune system in patients with arterial hypertension is already promoted and as such, it might represent the mechanisms through which arterial hypertension could be associated with severe forms of covid-19 infection. this is an area which requires further research. seemedj 2022, vol 6, no 1 arterial hypertension, risk of mortality and covid-19 77 southeastern european medical journal, 2022; 6(1) is the interaction of covid-19 infection and ras important for the clinical course of the disease? table 1. publications related to the association between severe covid-19 and arterial hypertension angiotensin-converting enzyme 2 (ace2) is a protein which has a crucial role in the entry of the covid-19 virus into the cells. it is the key matter of debate on the benefits or dangers of ras blockade in covid-19 infection. verdechia et al. labelled covid-19 an ace2-centric infectious disease (20). recently, the results of a study that used human cardiomyocytes as a model for researching the entry of sars-cov-2 into the cells have been published. after the viral infection, there is an increase in the synthesis of ace2, type 1 receptors for angiotensin ii, but no change in the synthesis of the angiotensinconverting enzyme (ace) (21). the same group of researchers have observed that the synthesis of ace2 in ace2-knockout (ace2-ko) cardiomyocytes was almost immeasurable, and, after the infection, these cells also had a negligible quantity of sars-cov-2. furthermore, authors findings shi et al. [10] cardiac injury is a common condition among hospitalised patients with covid-19 in wuhan, china and it is associated with a higher risk of in-hospital mortality. a total of 82 patients (19.7%) had cardiac injury. compared with patients without cardiac injury, these patients were older and had more comorbidities (e.g. hypertension in 49 of 82 [59.8%] vs 78 of 334 [23.4%]; p < .001). wu c et al. [11] arterial hypertension, especially poorly controlled hypertension, may play an important role in the severity of covid-19. guo t et al. [12] this retrospective single-centre case series analysed patients with covid-19 in wuhan, china, from 23 january 2020 to 23 february 2020. myocardial injury is significantly associated with a fatal outcome of covid-19, while the prognosis for patients with underlying cvd, but without myocardial injury is relatively favourable. 66 patients (35.3%) had underlying cvd, including hypertension, coronary heart disease and cardiomyopathy. dessie gz et zewotir t. [13] chronic comorbidities, complications and demographic variables, including acute kidney injury, copd, diabetes, hypertension, cvd, cancer, increased d-dimer, male gender, older age, current smoker and obesity are clinical risk factors for a fatal outcome associated with coronavirus. mubarik s et al. [14] prevalence of hypertension in 1,833 studied covid-19 patients was 40.5%. hypertension was associated with the severity and mortality of covid-19 infection. du y et al. [15] evidence from this meta-analysis suggested that hypertension was independently associated with a significantly increased risk of critical covid-19 and in-hospital mortality of covid-19. guan wj et al. [16] advanced age was associated with a higher prevalence of other comorbidities such as diabetes, renal impairment, arterial hypertension and obesity, which altogether increased the proportion of hypertensive patients. seemedj 2022, vol 6, no 1 arterial hypertension, risk of mortality and covid-19 78 southeastern european medical journal, 2022; 6(1) the authors treated human cardiomyocytes and human endothelial cells either only with the virus or with the virus combined with lisinopril or losartan. the amount of the virus was not increased in endothelial cells or in cardiomyocytes after the use of ace-inhibitors or sartans and the expression of proteins (ace2) did not significantly change after the use of these medications. this research provides an additional confirmation that ace-inhibitors or sartans do not promote sars-cov-2 infection. the hypothesis that advocated a negative role of ras blockade in sars-cov-2 infection was based on the fact that ras blockers, by inducing an enhanced ace2 expression, facilitate the entry of the virus into the cell (22). supporters of the opposing hypothesis, however, believe that the enhanced ace2 expression, along with ras blockade, facilitates increased synthesis of angiotensin 1-7, promoting its beneficial antiinflammatory and antifibrotic effects and preventing lung injury (figure 1) (23). figure 1. association between sars-cov-2 and renin angiotensin system ace – angiotensin-converting enzyme, acei – angiotensin-converting enzyme inhibitor, ang – angiotensin, arb – angiotensin type ii receptor blocker, at1r – angiotensin ii type 1 receptor, at2r – angiotensin ii type 2 receptor it has already been mentioned that ras blockers have a positive effect on the clinical course of pulmonary diseases without covid-19 infection (5,24). the significance of the beneficial impact of ace2 and angiotensin 1-7 on covid-19 infection is evident from the fact that, among other drugs which are synthesised for the treatment of covid-19 infection, scientists have been intensively working on recombinant ace2 and angiotensin 1-7 (20). the results of basic research are bringing us back to the epidemiological clinical research by mancia et al., which proved in a group of patients with confirmed covid-19 infection that neither ace-inhibitors nor sartans were associated with a higher probability of covid-19 infection (8). this was also confirmed in a meta-analysis by kurdi et al., which involved 72,372 patients from 27 studies (25). analysing a group of 1,002 patients with severe covid-19 infection, reynolds et al. concluded that ras blockade is not associated with severe forms of covid-19 infection (26). those data were confirmed in the previously mentioned meta-analysis by kurdi et al., where no association was observed between ras blockade and severe forms of pneumonia (25). the hope covid-19 registry, zhang et al. and research by gao et al. indicated a lower mortality rate in patients treated with ras seemedj 2022, vol 6, no 1 arterial hypertension, risk of mortality and covid-19 79 southeastern european medical journal, 2022; 6(1) blockers in comparison to those who did not receive these medications (18,27,17). finally, the meta-analysis by kurdi et al. also showed that the use of ras blockers was associated with a lower mortality rate and a lower risk of admission to intensive care units (25). another meta-analysis was published in 2021 and it included 101,949 patients from 52 studies, 26% of whom were treated with ras blockade (28). in this meta-analysis, the authors did not find an association between the use of ras blockers and higher mortality. on the contrary, they found that ras blockade has a positive effect, i.e. there were less deaths and severe complications after adjusting for all risk factors. in an extensive observational research, semenzato et al. noticed that patients treated with ace-inhibitors or sartans for a very long time had a lower risk of covid-19 infection than those treated with calcium channel blockers (29). this finding corresponds to the previously published results, according to which the use of ace-inhibitors or sartans increases survival, i.e. reduces mortality rates in hypertensive patients with covid-19 infection (30). the authors have not found any differences between aceinhibitors and sartans. however, a group of authors has recently raised an intriguing question whether sartans are the drug of choice for the treatment of arterial hypertension, and moreover, during covid-19 infection (31). there is obviously no hard evidence for this approach and further research is needed to either confirm or reject this premise. nevertheless, it is a good example of how the paradigm has switched from the fear of possible danger of applying ras blockers in patients with covid-19 infection to completely the opposite – exploring the details of their benefits. however, as mentioned, apart from sartans that obviously precede ace inhibitors, recombinant ace2 and angiotensin 1-7 are potential treatment options that are currently being explored (20,32). the real consequences of covid-19 infection for patients with arterial hypertension inadequate access to medical care, either due to the fear of infection or the medical personnel’s preoccupation with an enormous number of covid-19 cases, will lead to numerous negative impacts. the real consequence of covid-19 infection is a decline in the control of treated hypertensive patients, decline in adherence to medications, irregular blood pressure measurements and an increase in morbidity and mortality from cardiovascular, cerebrovascular and renal causes. instead of an isolated pandemic, a syndemic, clustering covid-19 and the excess burden of cardiovascular, cerebrovascular and kidney disease/deaths will become our harsh reality. conclusion in our opinion, the answer to the first question – does arterial hypertension increase the risk of covid-19 infection? – is most probably not, although there has been insufficient evidence so far. the answer to the second question – does arterial hypertension increase the risk of severe infections? – might be that it does, but only if arterial hypertension is not controlled well and treated properly. if it is controlled poorly, then there is a higher possibility of a fatal or more severe cytokine storm due to the chronic latent stimulation of the immune system among patients with arterial hypertension. the answer to the third question – is the risk of severe covid-19 infection associated with the use of drugs that block ras? – is that there is no evidence to suggest so. finally, are ras blockers beneficial or harmful? according to the recent studies, which indicate that patients treated with ras blockers might have a better prognosis, ras blockers might be beneficial. therefore, patients with stable arterial hypertension must continue taking ras blockers. however, in severe covid-19 infections accompanied by hypotension, patients must stop using ras blockers as well as any other antihypertensive drugs. numerous questions related to these issues are still left unanswered. as a stimulus for further considerations, we decided to select these two questions: should we introduce ras blockers for seemedj 2022, vol 6, no 1 arterial hypertension, risk of mortality and covid-19 80 southeastern european medical journal, 2022; 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28:129-139. doi: 10.1007/s40292-021-00439-9. author contribution. acquisition of data: bn, km, gi, jj, pv, ja, ks, bjn , jb administrative, technical or logistic support: bn, km, gi, jj, pv, ja, ks, bjn , jb analysis and interpretation of data: bn, km, gi, jj, pv, ja, ks, bjn , jb conception and design: bn, km, gi, jj, pv, ja, ks, bjn , jb critical revision of the article for important intellectual content: bn, km, gi, jj, pv, ja, ks, bjn , jb drafting of the article: bn, km, gi, jj, pv, ja, ks, bjn , jb final approval of the article: bn, km, gi, jj, pv, ja, ks, bjn , jb guarantor of the study: bn, km, gi, jj, pv, ja, ks, bjn , jb obtaining funding: bn, km, gi, jj, pv, ja, ks, bjn , jb provision of study materials and patients: bn, km, gi, jj, pv, ja, ks, bjn , jb statistical expertise: bn, km, gi, jj, pv, ja, ks, bjn , jb seemedj 2021, vol 5, no. 1 proliferation index (ki67) and immunophenotypes in invasive breast cancer 157 southeastern european medical journal, 2021; 5(1) original article differences in the value of proliferation index (ki67) and immunophenotypes between invasive breast cancers with respect to the axillary lymph node status 1 mirna kovačević 1, ivan švagelj 2*, mirta vučko 2, dražen švagelj 2,3 1 general county hospital vinkovci 2 department of pathology and cytology of the general county hospital vinkovci 3 department of pathological anatomy and forensic medicine, faculty of medicine osijek corresponding author: ivan švagelj, svagelj.ivan@gmail.com received: nov 26, 2020; revised version accepted: jan 25, 2021; published: apr 28, 2021 keywords: breast cancer; proliferation, immunophenotype, axillary, lymph node abstract introduction: present study aimed to determine the frequency of invasive breast cancer (ibc) immunophenotypes in the general county hospital vinkovci, examine a difference between the ages of patients with respect to immunophenotypes and axillary lymph node (aln) status, and determine differences in the frequency of positive alns with respect to immunophenotypes and the proliferation index (ki67), regardless of the immunophenotype. materials and methods: a monocentre cross-sectional study which included 289 patients diagnosed with invasive breast cancer was conducted in the period from 1 january 2011 to 31 december 2018. the expression of ibc biomarkers was determined by immunohistochemistry. results: the most common immunophenotype (41.54 %) was luminal b-like her2-negative (lumb/her2-). the mean age was 65.24 (± 12.04), with no age difference with respect to immunophenotypes (f = 0.64, p = 0.43) or aln status (t = 1.59; p = 0.11). a total of 167 patients (58 %) had their alns removed, 66 % of which were positive. lumb/her2appeared to have significantly more positive alns compared to the luminal a-like immunophenotype (p < 0.01), while a difference in the size of primary tumours between metastatic breast cancers of these two immunophenotypes has not been detected (p = 0.17). alns were more likely to be positive in those tumours with ki67 values higher than 20 % compared to the tumours in which ki67 was lower than or equal to 20 % (p < 0.01). conclusions: lumb/her2is the most prevalent ibc immunophenotype in patients in our institution and has significantly more positive alns compared to the luminal a-like immunophenotype. also, metastases to alns, regardless of the immunophenotype, are more common in patients with ki67 higher than 20 % than in those with ki67 lower than or equal to 20 %.. (kovačević m, švagelj i, vučko m, švagelj d. differences in the value of proliferation index (ki67) and immunophenotypes between invasive breast cancers with respect to the axillary lymph node status. seemedj 2021; 5(1); 157-169) seemedj 2021, vol 5, no. 1 proliferation index (ki67) and immunophenotypes in invasive breast cancer 158 southeastern european medical journal, 2021; 5(1) introduction breast cancer is the most common malignancy in women in the european union (1). breast cancers are divided into non-invasive (in situ), microinvasive and invasive carcinomas with recognisable subtypes. non-invasive carcinomas, unlike invasive carcinomas, do not have the ability to invade blood and lymphatic vessels and their tumour cells are limited to ducts. non-invasive carcinomas include ductal carcinoma in situ (dcis), intraductal papillary carcinoma, and lobular carcinoma in situ (lcis) (2). histologically, invasive carcinomas can be divided into ductal (80%) and lobular (10%), while the rest are special forms of breast cancer (paget's disease of the nipple, colloid mucinous carcinoma, tubular carcinoma, and solid papillary carcinoma with invasion) (3). according to literature, different types of breast cancers originate from the luminal and basal cells of the terminal duct lobular unit epithelium and they form a heterogeneous group of cancers that cannot be distinguished by histology (4, 8). when it comes to breast cancer treatment, monitoring and survival rates, molecular subtypes determined by microarrays with more than 2.000 genes take precedence over the standard histological phenotype. this method is not easily attainable and it is quite costly, so it is generally accepted to use the immunophenotypic classification instead, as determined by the immunohistochemical expression of oestrogen receptors (er), progesterone receptors (pr), proliferation index (ki67) and amplification of the human epidermal growth factor receptor 2 (her2) (2, 5). according to the new guidelines (croatian association for cancer research, 2018), er and pr are considered weak prognostic and strong predictive factors because hormone receptorpositive cancers have a better survival rate, since they predictively indicate a potential response to hormonal therapy (5-6). er indicates a response to cancer treatment via hormonal therapy such as tamoxifen, which blocks the growth of oestrogen-stimulated cancers, and a response to aromatase inhibitors that suppress oestrogen production (5). the next immunophenotypic marker, her2, is associated with a lower survival rate and is also a predictive factor. one of the main reasons for determining the her2 status is the identification of candidates for targeted anti-her2 therapy (trastuzumab, lapatinib, pertuzumab, and trastuzumab emtansine) related to her2positive breast cancers. the most controversial marker used is ki67, as it is determined in hotspot areas, and the result often depends on the quality of tissue fixation, formulation, and the type of antibody used (5). as a rule, a more distinct expression is associated with a lower survival rate, both in general and after the administration of neoadjuvant therapy (2, 5, 7, 8). in addition to being connected with immunophenotypes, the survival rate and treatment also depend on the penetration of malignant cells into blood and lymphatic vessels, tumour necrosis, age of the patient (younger age is a negative prognostic factor), size of the tumour, status of surgical margins and lymph nodes (2, 5). studies from different countries show a different prevalence of respective immunophenotypes of breast cancer, which points not only to a number of factors that may play a role in carcinogenesis, but also to possible differences due to nonstandardised protocols in pathohistological laboratories. in addition, the diagnosis of the correct immunophenotype is the cornerstone of proper treatment. therefore, it is necessary to evaluate one’s own work, among other things, comparing it with other laboratories in croatia and other countries in order to detect possible problems in time and improve or at least maintain quality at an acceptable level. therefore, the objective of this research was to determine the overall incidence of breast cancer and individual immunophenotypes in general county hospital vinkovci (cbc), to examine whether there is a difference between the age of women diagnosed with cancer related to immunophenotypes and lymph node status, and to determine the differences in the frequency of positive axillary lymph nodes with respect to immunophenotypes and especially with regard to high ki67 values, regardless of the immunophenotype. seemedj 2021, vol 5, no. 1 proliferation index (ki67) and immunophenotypes in invasive breast cancer 159 southeastern european medical journal, 2021; 5(1) materials and methods study structure and materials the paper is based on a monocentre crosssectional study on historical data (9). the findings of patients diagnosed with breast cancer in the period from 1 january 2011 to 31 december 2018 were collected from the archives of the department of pathology and cytology of the general county hospital vinkovci. from a total of 296 patients diagnosed with breast cancer, three were men, who were excluded from further processing. also, because of the criteria of invasiveness, four female patients diagnosed with carcinoma in situ were excluded. therefore, the final sample consisted of medical findings of 289 female patients with said diagnosis. the research was approved by the ethics committee of the general county hospital vinkovci. methods data of interest in the findings expressed er, pr, her2, and ki67 and were obtained using routinely prepared histological samples, formalin fixed paraffin embedded – ffpe, and finally immunohistochemically stained. the preparations were stained with dako envision flex kit by treating the sample with peroxidase blocking reagent (for 5 minutes), rinsed with envision flex wash buffer and treated with the primary antibody called flex monoclonal rabbit anti-human estrogen receptor α, clone ep1 (dako), or other antibodies like mouse antihuman progesterone receptor, clone pgr 636 (dako), flex monoclonal rabbit anti-human ki67 antigen, clone mib-1 (dako) and monoclonal rabbit anti-human her 2 protein (dako) (for 20 min), rinsed with envision flex wash buffer and stained with envision flex hematoxylin. finally, the preparations were covered with sakura tissue-tek film in an automatic glass coating device sakura tissuetek film (10). analysis and interpretation of the immunohistochemical staining findings were carried out according to the who guidelines (8). the er and pr findings are considered positive if 1 % or more tumour cells show immunohistochemical nuclear positivity, and negative in case of absence of nuclear positivity or strong nuclear positivity in less than 1 % of total tumour tissue. if tumour cells show absence of membrane positivity or very weak membrane positivity to the her2 antibody, the her2 status is indicated with 0 or 1+ and the finding is considered negative. in contrast, if more than 10 % of tumour cells have membrane positivity, the finding is considered positive and marked with 3+. when tumour cells show incomplete membrane positivity in 10 % of tumour tissue or if the positivity is at the borderline of 10 % of the total tumour tissue, the her2 status is denoted by 2+. in this case, additional in situ hybridisation (fish/cish) is required to determine the existence of amplification of the her2 gene and categorise the tumour as her2-positive or her2-negative (8). according to immunohistochemically determined expression, we distinguish the following five immunophenotypes: luminal alike, luminal b-like her2-negative, luminal b-like her2-positive, her2-positive and triplenegative breast cancer (table 1). luminal a-like immunophenotype has the best survival rate, while a triple-negative immunophenotype has the worst survival rate (2). luminal b-like her2negative breast cancer immunophenotype should exhibit higher ki67 values than luminal alike. both immunophenotypes are her2negative and er-positive, but the luminal b-like her2-negative immunophenotype has a lower survival rate, so the separation of these two breast cancer immunophenotypes is essential for therapy prescription. according to the recommendations of the st. gallen oncology conferences from 2013 and the 2017 croatian guidelines for quality assurance in breast cancer screening and diagnosis, the cut-off value for this factor is 20 % (11-12). the cut-off value is important in clinical practice because it indicates which patients should receive a more intensive therapy (13). seemedj 2021, vol 5, no. 1 proliferation index (ki67) and immunophenotypes in invasive breast cancer 160 southeastern european medical journal, 2021; 5(1) table 1. immunophenotypes of breast cancer immunophenotype er pr her2 ki67 luminal a-like + + < 20 % luminal b-like her2negative* + or low* > 20 %* luminal b-like her2positive + +/+ +/ her2-positive + +/ triple-negative +/ *at least one of the following criteria is required after thorough processing, the pathohistological findings indicate tumour size, malignancy, ki67, hormone receptor status, her2 status, tumourto-resection-margin ratio, number of examined and positive axillary lymph nodes, and tumourto-blood and lymphatic vessel ratio. finally, after processing these data according to the tnm staging system, the stage of breast cancer is determined indicating the probability of being cured (14). statistical methods the variables were collected in ms excel and processed in the program r (15). the sample was described using descriptive statistical methods. the shapiro–wilk w-test (or, in case of a large number of samples, the kolmogorov–smirnov test) was used to examine the distribution of numerical variables. a comparison of categorical variables was carried out using the pearson's chi-squared test and the fisher's exact test. anova was used when comparing several groups of numerical variables, given the distribution of data was normal, and an adequate post-hoc test was used in cases of statistically significant results. the parametric student’s ttest was used to compare two groups of numerical variables with normal distribution. in this paper, the level of statistical significance for all tests used for comparisons was defined at p < 0.05. results the research included 289 patients with breast cancer. the highest number of patients (45) was in 2018, and the lowest (26) in 2011 (χ2 = 5.09, p = 0.65). a difference was observed in the frequency of occurrence of certain immunophenotypes throughout the research period: the luminal b-like her2-negative immunophenotype was more common in 2013 than in other years (χ2 = 3.41, p = 0.03), while the luminal b-like her2-positive immunophenotype was prevalent in 2017 (χ2 = 3.89, p < 0.01) (figure 1). according to the pathohistological diagnosis, the majority of patients (238) were diagnosed with invasive ductal carcinomas. invasive lobular breast cancers were present in 30 patients and invasive mucinous carcinomas in 10. the rest of the patients had less common pathohistological types of breast cancer (table 2). seemedj 2021, vol 5, no. 1 proliferation index (ki67) and immunophenotypes in invasive breast cancer 161 southeastern european medical journal, 2021; 5(1) figure 1. distribution of patients with breast cancer with regard to immunophenotypes by years during the research period luminal b-like her2-negative immunophenotype was more common in 2013 than in other years (*χ2 = 3.41, p = 0.03), while the luminal b-like her2-positive immunophenotype was most common in 2017 (# χ2 = 3.89, p < 0.01). when it comes to other immunophenotypes, there was no difference in the frequency between the years within the research period (figure 1). index: luma = luminal a-like; lumb = luminal b-like her2-negative; lumb/her2 = luminal b-like her2positive; her2 = her2-positive, tr_neg = triple-negative table 2. pathohistological diagnosis of breast cancer (n (%)) histological type of breast cancer number (%) of patients infiltrating duct carcinoma nos 238 (82.3) lobular carcinoma nos 30 (10.4) mucinous adenocarcinoma 10 (3.5) solid papillary carcinoma with invasion 5 (1.7) metaplastic carcinoma nos 3 (1.0) tubular carcinoma 2 (0.7) apocrine adenocarcinoma 1 (0.4) total 289 (100) seemedj 2021, vol 5, no. 1 proliferation index (ki67) and immunophenotypes in invasive breast cancer 162 southeastern european medical journal, 2021; 5(1) the mean age of the patients was 65.24 (95 % ci 63.84 – 66.63). the sample follows the normal distribution (d = 0.05; p = 0.4). regarding the immunophenotype of breast cancer, there was no difference in the age of the diseased patients (f = 0.64, p = 0.43) (figure 2).. figure 2. age of patients with breast cancer with respect to immunophenotypes the shapiro-wilk test helped determine that the age of all patients with respect to the immunophenotypes was distributed according to the normal distribution. there was no difference in the age of patients with regard to the breast cancer immunophenotype (f = 0.47, p = 0.49). the red line indicates the mean age of all patients, which is 65.24 (figure 2). index: luma = luminal a-like; lumb = luminal b-like her2-negative; lumb/her2 = luminal b-like her2-positive; her2 = her2-positive; tr_neg = triple-negative. axillary resections were performed in our institution on a total of 167 (58 %) patients. of these 167 patients, 111 (66 %) had positive axillary lymph nodes (proven metastases of cancer tissue), and 56 (34 %) had negative results. the difference between respective immunophenotypes was determined with regard to the presence of positive axillary lymph nodes. the luminal b-like her2-negative immunophenotype has positive axillary lymph nodes significantly more frequently than the luminal a-like (p < 0.01) (figure 3). however, no difference in the primary tumour size (relative to pt) was observed between these immunophenotypes with positive axillary lymph nodes (p = 0.17) (table 3). furthermore, no age difference was observed between patients with positive and those with negative axillary lymph nodes (t = 1.59; p = 0.11). patients with a ki67 value higher than 20 % were more likely to have positive axillary lymph nodes than those with a ki67 value lower than or equal to 20 %, regardless of the immunophenotype (χ2 = 9.26, p < 0.01) (figure 4).. seemedj 2021, vol 5, no. 1 proliferation index (ki67) and immunophenotypes in invasive breast cancer 163 southeastern european medical journal, 2021; 5(1) figure 3. incidence of positive axillary lymph nodes with respect to immunophenotypes of breast cancer a difference was observed between the immunophenotypes of breast cancer in the frequency of detected positive axillary lymph nodes (χ2 = 12.87, p < 0.05), and a post-hoc analysis found that the luminal b-like immunophenotype has positive axillary lymph nodes much more frequently compared to the luminal a-like immunophenotype (p < 0.01) (figure 3). index: luma = luminal a-like; lumb = luminal b-like her2-negative; lumb/her2 = luminal b-like her2-positive; her2 = her2 positive; tr_neg = triple-negative; ln+ = positive axillary lymph nodes; ln= negative axillary lymph nodes. figure 4. number of patients with positive and negative axillary lymph nodes depending on the level of the proliferation index (ki67) positive axillary lymph nodes are more common in breast cancer patients with ki67 higher than 20 % (ki67 > 20%) compared to those with ki67 lower than or equal to 20 % (ki67 ≤ 20 %), regardless of the immunophenotype in question (χ2 = 9.26, p < 0.01) (figure 4). index: ln+ = positive axillary lymph nodes; ln= negative axillary lymph nodes seemedj 2021, vol 5, no. 1 proliferation index (ki67) and immunophenotypes in invasive breast cancer 164 southeastern european medical journal, 2021; 5(1) table 3. primary tumour size in luminal a-like and luminal b-like her2-negative carcinomas that have positive axillary lymph nodes. primary tumour size immunophenotype pt1 pt2 pt3 pt4 total lum a 10 7 1 2 20 lum b/her217 26 10 2 55 total 27 33 11 4 75 there was no difference in the size of the primary tumour between the luminal a-like and luminal b-like her2-negative immunophenotypes of breast cancers with positive axillary lymph nodes (p = 0.17). index: luma = luminal a-like; lumb/her2 = luminal b-like her2-negative discussion this research has shown that the luminal b-like her2-negative immunophenotype is the most common immunophenotype of breast cancer in patients who have undergone surgery in our facility. in addition, positive axillary lymph nodes are more common in case of this immunophenotype than the luminal a-like immunophenotype, regardless of the size of the primary tumour. by way of explanation, due to the known correlation between tumour size and metastases of breast cancer to axillary lymph nodes (8), the observed difference in the incidence of metastatic breast cancers regarding these immunophenotypes was further analysed with respect to primary tumour size (pt). although the difference in the size of the primary tumour subject to examination has not been confirmed, it should be emphasised that the possibility that this could be determined by research on a larger number of samples cannot be ruled out. also, this paper indicates that metastases to axillary lymph nodes, regardless of the immunophenotype, are more common in patients with breast cancer with a ki67 value higher than 20 % than in those with a ki67 value lower than or equal to 20 %. the analysis of 289 patients with breast cancer found that the most common histological type was invasive duct carcinoma (82.3 %), followed by invasive lobular carcinoma (10 %) and invasive mucinous carcinoma (3.5 %). the same order of histological subtypes can be found in studies conducted in germany (16), saudi arabia (17), pakistan (18) and nigeria (19), while the invasive ductal type was slightly less common (59 %) in one italian study, and it was followed by invasive lobular type with 14 % (20). on the other hand, invasive ductal carcinoma is the most common in china (94 %), but it is followed by invasive mucinous carcinoma, with invasive lobular carcinoma in the last place (21). the mean age of the patients included in this research was 65.24 years, which is similar to the results in clinical hospital centres, general hospitals, clinics and polyclinics all across the republic of croatia (22), as well as serbia (65.59 ± 10.17) (23). a slightly lower mean age (55-57) was detected in studies carried out in germany (16), japan (24) and brazil (25), while the lowest mean age (43-48) was in sweden (26), nigeria (19) and pakistan (18) (table 3). the significantly lower age of breast cancer patients in africa is explained by nigerian authors by the thesis that breast cancer occurs up to 15 years earlier in black people (19). an almost equal representation of respective immunophenotypes as found in our sample was noted in another research conducted in croatia (22), and in a country bordering croatia – serbia (27), but also in sweden (26) (table 4). the luminal a-like immunophenotype is most prevalent in certain european countries (germany and italy) (16, 20), asia (china and japan) (21, 24), as well as in saudi arabia and the united states (17, 28). it is interesting to single out countries such as pakistan (18), vietnam (26), and especially nigeria (19), in which her2-positive and triple-negative immunophenotypes that are more common in younger people, are more aggressive and have a lower survival rate, seemedj 2021, vol 5, no. 1 proliferation index (ki67) and immunophenotypes in invasive breast cancer 165 southeastern european medical journal, 2021; 5(1) prevail partially or predominantly (29). this is a possible explanation as to why these three countries also have slightly lower age statistics when it comes to patients with breast cancer (table 4). in addition to the presence of said immunophenotypes, the age of patients is certainly affected by the implementation of prevention programs, which is evident in the example of sweden, which, despite the distribution of immunophenotypes similar to that in our research, has a much lower median age. in sweden, the age threshold of women included in the prevention program is 40, i.e. 10 years younger than in the republic of croatia, where the threshold is 50 (30, 12). the observed geographical grouping of certain immunophenotypes suggests that, in addition to genetic, a certain significant role in the carcinogenesis of breast cancer is also played by environmental factors, which should certainly be investigated in future research. when it comes to the frequency of positive axillary lymph nodes, results from our sample made it clear that the luminal b-like immunophenotype metastasises significantly more frequently to axillary lymph nodes compared to the luminal a-like immunophenotype. a group of authors from serbia obtained similar results (27). contrary to our result, the authors of studies from italy (20) and brazil (25) did not come across significant differences. however, it should be pointed out that a general limitation of these comparisons with other studies lies in the fact that few of them have used a slightly different methodology in the determining of breast cancer immunophenotypes (indicated in table 4). minor differences in the methodology of the first group of studies is a consequence of a change in the cut-off value of the proliferation index from 14% to 20%, accepted after the conclusions of the st. gallen oncology conferences in 2013 (11). in our opinion, although these important differences in determining the proliferation index have a huge impact on the type of treatment of each individual patient with breast cancer, they have a minor effect on the total immunophenotype ratio because a small minority of cases could be reclassified into a different immunophenotype if another classification system (the same as in our study) was used. on the other hand, major differences in the methodology presented in the second group of studies are the result of a lack of use of the proliferation index, which can be seen from the absence of the luminal b-like her2-negative immunophenotype category (table 4). therefore, the previously discussed comparison of our results with the results from these studies could be biased. regardless of the immunophenotype, in our research, axillary lymph nodes were more frequently positive in those patients with a ki67 value higher than 20 %. a group of authors from serbia obtained a similar result, indicating the connection between positive axillary lymph nodes and elevated ki67, but, in contrast to our study, it had to do with tumours in which ki67 was higher than 14 % (27). in a research conducted in turkey, it was found that patients with higher stages of positive lymph nodes (pn2 and pn3) have a higher average level of ki67 expression than those with lower stages and negative lymph nodes (pn0 and pn1) (31). contrary to these conclusions, a research in ethiopia found that there was no difference in the level of the ki67 proliferation index between breast cancers that have positive and negative axillary lymph nodes, regardless of whether all cancers were divided into three groups according to the ki67 level (ki67 < 15 %; 15 % < ki67 < 30 %; ki67 > 30 %) or the average level of the proliferation index (13). since the level of the ki67 proliferation index amounting to 20 % is the limit that groups certain breast cancers into luminal a-like and luminal b-like immunophenotypes, the result obtained indirectly confirms that the luminal b-like immunophenotype has a lower survival rate than the luminal a-like immunophenotype, which is consistent with the literature (11). seemedj 2021, vol 5, no. 1 proliferation index (ki67) and immunophenotypes in invasive breast cancer 166 southeastern european medical journal, 2021; 5(1) table 4. frequency (%) of immunophenotypes in different countries of the world country sample size age (in years) luma (%) lumb (%) lumb/her2 (%) her2 (%) tr_neg (%) reference germany 4102 57 44.7 31.8 6.2 5.0 12.3 (16)# japan 363 56.7 30.6 26.2 19.0 11.3 12.9 (24)# brazil 269 55.4 23.79 10 34.61 14.50 17.10 (25) # serbia 108 61 25.92 47.22 19.44 1.85 5.55 (27) # italy 1487 -* 34.09 25.21 11.49 10.15 19.03 (20) # china 3198 51 65.3 19.0 6.5 9.2 (21) § saudi arabia 359 49.8 58.5 14.5 12.3 14.8 (17) § nigeria 118 46.9 28.8 6.7 17.9 46.6 (19)§ pakistan 285 43.3 21.05 48.77 18.4 11.22 (18) § usa 50 571 -* 72.7 10.3 4.6 12.2 (28) § vietnam 237 47.7 10.6 33.5 23.0 19.3 13.6 (26) sweden 237 51.3 31.6 41.3 8.9 7.6 10.6 (26) croatia 1868 62.3 31.32 45.67 11.67 4.50 6.80 (22) croatia 289 65.24 26.64 41.54 15.22 6.22 10.38 this research * the data on patients’ age are not expressed as mean or median. # cut-off value of proliferation index is 14% § proliferation index was not used luma = luminal a-like; lumb = luminal b-like her2-negative; lumb/her2 = luminal b-like her2-positive; her2 = her2positive; tr_neg = triple negative however, it is important to point out one of the limitations of these comparisons. in clinical pathology, the above-mentioned immunohistochemical marker ki67 is observed by a microscope, using a semiquantitative method, the interpretation of which is subject to numerous factors, such as the experience of the pathologist, quality of equipment, and quality of sample processing. therefore, that may be the cause of interlaboratory discrepancies in the interpretation of samples, and therefore lead to different results. this paper has potentially useful clinical as well as public health implications. primarily, this study describes the distribution and characteristics of breast cancer, which may be useful in planning, adjusting, and improving treatment options, but also in assessing the risk of disease recurrence and death depending on the immunophenotype of breast cancer. in addition, a slight but seemedj 2021, vol 5, no. 1 proliferation index (ki67) and immunophenotypes in invasive breast cancer 167 southeastern european medical journal, 2021; 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revised version accepted: april 20, 2017; published: april 24. 2017 keywords: gestational diabetes, 3d power doppler, histopathology, umbilical cord abstract aim: the resistance of placental blood vessels might be increased in diabetic pregnancies. this increased resistance can affect uteroplacental blood flow and influence the oxygen and nutrient supply of the fetus and fetal growth. our aim was to compare the ultrasonographic, pathomorphologic data and vasoreactivity of umbilical and placental vessels of gestational diabetic newborns with that of normal pregnancy newborns. methods: in this case-control study the placental vascularization of pregnant women was determined by 3d power doppler ultrasound technique. we calculated the vascularization index (vi), flow index (fi) and vascularization flow index (vfi). we performed a tissue bath experiment (treatment with oxytocin and desmopressin) on umbilical vessels and collected pathomorphologic data according to the royal college of pathologists’ 2011 protocol. results: the placental vascularization index and the umbilical artery s/d were significantly lower in the case group. the mean vi was 6.21% (±2.69 sd) in control versus 3.5% (±2.97 sd) (p<0.05) in gdm. the mean value of the umbilical artery s/d was 2.27 (±0.22 sd) and 2.18 (±0.45 sd) (p<0.05) respectively. in an isolated tissue bath experiment, oxytocin and desmopressin did not elicit significant contraction on umbilical cord vessels. conclusion: our results suggest that umbilical vessels might have a different receptor pathway regulation that can compensate for the rheological changes in the pregnant woman’s blood flow and gives opportunity for selective therapy to fetuses more vulnerable to hypoxia. (jakó m, surányi a, kaizer l, domokos d, bártfai g. the correlation of ultrasonographic and pathophysiologic measurements of umbilical vessels in gestational diabetes. seemedj 2017;1(1);40-49) seemedj 2017, vol 1, no. 1 the correlation of ultrasonographic and pathophysiologic measurements… 41 southeastern european medical journal, vol 1, 2017. introduction gestational diabetes mellitus (gdm) affects 14% of pregnancies, or 135,000 women a year in the usä, although its incidence varies nationwide. the mother’s risk for conversion to type 2 diabetes ranges from 2.6 to 70% over a period from 6 weeks to 28 years postpartum (1). there are many, noncorresponding data regarding the prevalence of gdm in hungary. in a populationbased screening program, 75 g ogtts were offered to all pregnant women between 24–28 weeks of gestation and evaluated according to who criteria. in that study 8.7% of pregnant women were diagnosed with gdm, and the risk increased linearly with maternal age. women with the highest bmi (≥ 29.2 kg/m2) had decreased risk compared to women with a bmi of 26.1–29.1 kg/m2 (p<0.05). these results suggest that pre-pregnancy bmi and previous deliveries elevate the risk of gdm only to a certain level, above which the risk decreases (2). postprandial glucose concentrations steadily increase as tissue sensitivity to insulin decreases. to maintain proper blood glucose value along pregnancy, the pancreatic gland of the mother increases insulin secretion to compensate. insulin resistance and impaired insulin secretion by the pancreas cause the development of gdm, especially during the third trimester of pregnancy (3). pregnancy is a complex metabolic state that involves dramatic alterations in the hormonal milieu, changes in adipokines and inflammatory cytokines. there are high levels of estrogen, progesterone, prolactin, cortisol, human chorionic gonadotropin, placental growth hormone, human placental lactogen, leptin, tnfα, and oxidative stress biomarkers. in addition, decreases in adiponectin worsen maternal insulin resistance in the second trimester (4). the placental and fetal demands for glucose are considerable and approach the equivalent of ~150 grams per day of glucose in the third trimester. glucose transport to the fetus occurs in direct proportion to maternal glucose levels, and is augmented by a five-fold increase in a placental glucose transporter, (glut-1) which increases transplacental glucose flux (5). fetuses exposed to a high glucose environment usually have medical complications after delivery, including infant respiratory distress syndrome, cardiomyopathy, hypoglycemia, hypocalcemia, hypomagnesaemia, polycythemia, and hyperviscosity. the hyperglycemia and adverse pregnancy outcome (hapo) study, which investigated about 25,000 pregnant women in 15 centers, found that even in subclinical hyperglycemia, higher maternal bmi was associated (odds ratio [95% confidence interval] with an increased frequency of birthweight >90th percentile (3.52 [2.48–5.00]) and percentage of body fat >90th percentile (3.28 [2.28–4.71]), caesarean section (2.23 [1.66–2.99]), and cord c-peptide >90th percentile (2.33 [1.58–3.43]) (6). the first line of management of women with gestational diabetes is medical nutrition therapy and a given minimum of exercise. patients who fail to maintain normal glycemic values via diet and exercise therapy receive insulin (7). as umbilical cord vessels represent a suitable model for the study of vascular alterations brought about by gdm, the aim of the present work was to compare the ultrasonographic vascular flow measurements to pathological microvascular changes, and also to test the vasoreactivity of the vessels. the chosen agents were oxytocin, which is present naturally at the time of pregnancy and can pass through the placenta (8), and vasopressin, an oxytocin receptor agonist. oxytocin and vasopressin are both peptide hormones, and oxytocin is widely used to promote labor contractions in clinical practice. the effects of these peptides are mediated via transmembrane receptors. both the oxytocin receptor (otr) and the v1a vasopressin receptor (v1ar) are expressed in human myometrium. the expression of otr is significantly higher in gravid uterus while v1ar expression is not significantly elevated compared to non-gravid uterus (9). oxytocin gene and receptor expression have also been shown in human chorion, deciduas and amnion seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 42 southeastern european medical journal, vol 1, 2017. epithelial cells with the onset of labor. the structural similarity of the two peptides (approximately 80%), as well as the homology of the receptors, causes some cross-reactivity between peptides and receptors (10).the placental presence of v1ar mrna is known in sheep but not in humans. the lack of avp mrna suggests the fetal origin of the ligand (11). pharmacological studies suggest the existence of vascular endothelial avp/ot receptors that mediate a vasodilatory effect. however, the nature of the receptor subtype(s) involved in this vasodilatory response remains controversial. rt-pcr experiments with total rna extracted from a human umbilical vein endothel amplified the ot receptor sequence only, but various vascular beds display different sensitivities to avp (12). other studies suggest that the vasopressine receptor type 2 is responsible for vasodilation via nitrogen-monoxide production (13,14). to the best of our knowledge, no receptor subtypes have been investigated in umbilical arteries, and the fetal production of the ligands is a hypothesis. we hypothesize that during myometrial contraction the above mentioned vascular relaxation can maintain fetoplacental circulation. the aim of this study was to investigate whether the vasodilatory effect of avp/ot receptor pathways is present in fetoplacental circulation. we also wanted to investigate whether the diabetic vasculopathy and vessel calcification affects this vasodilation. materials and methods ultrasound screening we collected samples of umbilical cords from the department of obstetrics and gynecology at the university of szeged from october, 2013 until june, 2014. focusing on the well-known risk factors, a detailed medical history was taken, and relying on the results of routine ultrasound screening, fetal biometry and anamnesis, we set up a case group of gestational diabetic pregnancies (gdm fetuses) (n = 5) and compared them to a control group (n = 22). gestational diabetes was diagnosed at gestational weeks 24-28 as hyperglycemia, first detected in pregnancies that met the who’s guide criteria (15). each of the patients with gestational diabetes was treated with dietary restrictions and monitoring. no insulin treated patients were included. the control group consisted of patients without diabetes or other endocrinologic, vascular or pulmonary disease. they delivered normal weight, healthy newborns. those who did not fit any of the above criteria were excluded from this study. gestational age was determined based on the first day of the last menstrual period and on ultrasound biometry (crown-rump length [crl] and biparietal diameter [bpd]) at the 10th week of pregnancy. all patients were scanned in a semirecumbent position. the factorial default setting "obstetrics/2-3 trimester" was used in 2d mode. the examination was followed by a fetal biometry to assess bpd, head circumference (hc), abdominal circumference (ac) and femur length (fl). fetal weight was calculated by the formula b of hadlock (16). a conventional color doppler study of umbilical arteries was also performed and the systolic velocity/diastolic velocity(s/d) ratio, the resistance index (ri) and pulsatility index (pi) were read from the ultrasound report display. the images used for the determination of placental volume and three-dimensional power doppler (3dpd) indices were obtained at the time of the visit. all 3-d scans and the 2-d ultrasound measurements were performed. voluson 730 expert ultrasound machines equipped with a multifrequency probe (2-5 mhz) were used to acquire all images (ge medical systems, kretztechnik gmbh&co ohg, austria). each sample was examined using a 3-d rendering mode, in which the color and gray value information was processed and combined to produce 3-d images (mode cent; smooth: 4/5; frq: low; quality: 16; density: 6; enhance: 16; balance: 150; filter: 2; actual power: 2 db; pulse repetition frequency: 0.9). a power doppler window (pulse repetition frequency at 900 hz and wall filter of 50 hz) was placed over the placenta mapping the vascular tree from the basal to chorionic plates. we used fast low seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 43 southeastern european medical journal, vol 1, 2017. resolution acquisition to avoid any kind of artifact. the 3-d static volume box was placed over the highest villous vascular density zone at umbilical cord insertion (17-19). during gestation we recorded one sample from each patient. the stored volumes were further analyzed using the virtual organ computer-aided analysis (vocal) program pertaining to the computer software 4d view (ge medical systems, austria, version 10.4) by an expert in 3-d analysis (18).the power doppler technique shows high sensitivity, because it is based on amplitude instead of mean frequencies to depict the vascular tree (19). moreover, the color mapping is independent from the angle of insonation and does not show “aliasing”. the placental vascular indices from sonobiopsy are a valid alternative for evaluation of the placental vascular tree when visualization of the entire placenta is not feasible. based on the ultrasonographic technique vascularization index (vi, the perfusion of the tissue, the percentage of blood flowing through), the flow index (fi, the amount of red blood cells flowing through a given area in a given time) and their combination, the vascularization flow index (vfi) can be determined (20). the spherical sample volume was consistently 28ml. the vocal program calculated automatically gray and color scale values in a histogram from the acquired spherical sample volume in each case (18-19). the delivery mode and duration with gestational age were recorded in the delivery room along with the body weight and body length of the newborn, of which rhohner's ponderal index could be calculated with a normal value between 2.2 and 2.9. for statistical reasons, the gender and 1-5 minute apgar-scores were also registered. tissue bath study at the placental insertion two 10 cm long segments of the umbilical cord were removed immediately and the one proximal to the henseleit solution (118 mm nacl; 4.7 mm kcl; 1.2 mm kh2po4; 1.2 mm mgso4.7h2o; 2.5 mm cacl2 2h2o; 25 mm nahco3, 11.7 mm dextrose). the samples were processed and stored in an ice-lined stainless steel thermos, and the solution was freshly prepared on a weekly basis and stored in a refrigerator until used. the vessels were dissected from wharton’s jelly within 24 hours to maintain their reactivity (11).the arteries were cut into two 3-5 mm long rings, and 4 similar rings were prepared from the vein. they were suspended on stainless steel hooks and placed in a tissue bath containing a krebs-henseleit solution at 37°c, exposed to 2g initial tension and bubbled with carbogen (95% o2, 5% co2). during the sixty-minute equilibration time we washed the system throughout with fresh solution every 15 minutes and let the spontaneous basal tone of the vessels to develop. oxytocin and desmopressin were added to the vessels in the concentration range of 10-9-1x10-5m and 10-11-10-7m, respectively. the change in the vascular tone was detected by isometric sensors and recorded and analyzed by isosys s.p.e.l. advanced computer software (experimetria ltd, budapest, hungary). the results are interpreted as the percentage of the basal, spontaneous vascular tone represented as 100% in value. the functionality of the vessel rings was tested with serotonin, which caused vasoconstriction, proving the viability of the vessels. pathological examination the placenta, along with the other segment of the cord, was stored in formalin for the morphological and histological examinations. the macroscopic evaluation of the samples followed the protocol published in 2011 by the royal society of pathologists (22). from the turns by the vessels, we calculated the umbilical coiling index (uci), with the standard normal value of one. in addition, with two scalpels fixed in the distance of two cm, three sections of the cord were cut. we stained their ends with ink and made prints on a millimeter paper, allowing them to be pushed to the paper by their natural weight. all the samples were handled by the same person. the area of the cross-sectional surface was measured using image j 1.47v (2013) computer software on 3x6 prints in each case. seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 44 southeastern european medical journal, vol 1, 2017. for the preparation and evaluation of the histological sections of the samples, we used the remaining cord and placental tissue. these data fundamentally determined the subsequent evaluation of the results. two-way anova statistical analysis was performed via prism 6 (c 1992-2012 graphpad software inc.). results all together 27 placentas and cords (22 control and 5 gdm) were examined. the mean age of the pregnant women in the control group was 32.09 (±5.66 sd) versus 34.20 (±4.97 sd) in the gdm group. the number of previous gestations was 1.46 in the control and 2.8 in the gdm group. we recorded two previous intrauterine growth restricted newborns in the diabetic group, and one macrosomic newborn in the control group. pregnancy has long been known to be diabetogenic in the sense of the progressive metabolic changes. proportional regression analysis revealed that the relative risk of developing non-insulin-dependent diabetes mellitus postpartum was 1.95 (95% ci, 1.63-2.33) for each 4.5 kg of weight gained during followup (23). in the control group, medications taken during pregnancy were either one of the available overthe-counter pregnancy multivitamins or none at all. in the gdm group, methyldopa, magnesium, vitamin b6, verapamil-hydrochloride, calciumdobezilat and multivitamins were take none month prior to giving birth. the clinical data of the pregnant women and their newborns are summarized in table 1. all three vessels were present in each case along the entire length of the three to seven days old formalin-fixed samples. the pathomorphological data are shown in table 2. the coiling of the cords is typically counterclockwise, twisted to the left, but twisting to the right occurred in 4 cases too; in the healthy control group (n = 3) and in the gdm group (n = 1). the uci was calculated as the number of full turns made by the vessels within five centimeters. perivascular hematoma and one hemangioma cavernosum were observed in the control, while two cases of multiple false knots, a slight funisitis and edematous cord were found in the gdm group. true knots, abnormal vascular division, stenosis, striction, meconium staining, maceration, thrombosis, calcification around the vessels, or other differences, discoloration, or odor were not observed. the cross-sectional surface area of the formalinfixed tissue was compared to the measurements made on the ultrasonographic images. the cross-sectional surface area of the gdm fetuses’ umbilical cords is roughly 65% of control cords measured in utero, and 76% after formalin fixation. the mean cord cross-sectional areas measured by 2d ultrasound were 344.00 mm2 (±0.46 sd) in the control group and 224.50 mm2 (±0.74 sd) in the diabetic group (p<0.05). after formalin fixation for three to seven days the cross-sectional areas were 124.00mm2 (±35.47 sd) and 93.75mm2 (±9.86 sd) respectively (p<0.05). measured with the ultrasonographic vocal technique, we found that the mean fi was 34.45% (±4.59 sd) in the control group and 29.80% (±3.68 sd) in the gdm group (ns); the mean vi was 6.21% (±2.69 sd) and 3.5% (±2.97 sd) (p<0.05), and the mean vfi was 2.31% (±1.23sd) and 0.95% (±1.20 sd) (ns) respectively. the mean value of the umbilical artery s/d was 2.27 (±0.22 sd) in controls and 2.18 (±0.45 sd) (p<0.05) in diabetic pregnancies. oxytocin did not elicit significant change in the vascular tone in the arteries and the veins, and the responsiveness of the vessels was not different significantly in the two groups (figure 1). desmopressin was also added to the vessels in logarithmic noncumulative concentrations (24) as shown in figure 2, but there was again no response. discussion in our study, umbilical 3d ultrasound examination showed that, compared to their gestational age, gdm fetuses have the normal s/d velocity curve. the vi is lower than in normal pregnancies because the development of blood vessels is not proportional to the growth of the placenta. in addition, they are damaged by the high serum glucose level. the seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 45 southeastern european medical journal, vol 1, 2017. increased entry of glucose into vascular smooth muscle cells via glut1 transporter mediated by il1β activates the pentose phosphate pathway, thus permitting some of the excess glucose to be metabolized via this route. this in turn led to an over activation nadph oxidase, resulting in increased generation of free radicals and the subsequent downstream proinflammatory signaling. free radicals, inflammation and oxidative stress lead to atherosclerosis (25). the uteroplacental circulation can be detected by the flowmetry of the umbilical and uterine arteries. the wave and shape of the flow, both in umbilical and uterine arteries, depends on the table 1. clinical and biometric data of the pregnants and newborns. control (n=22) gdm (n=5) mean sd sem mean sd sem p bmi before pregnancy 21.68 3.18 1.20 24.42 5.58 2.79 0.3968 bmi after pregnancy 25.85 1.85 0.58 30.83 4.44 1.98 0.0425* previous pregnancies 1.46 1.97 0.59 2.80 0.84 0.37 0.0226* systolic blood pressure (hgmm) 123.20 25.86 7.80 124.00 19.49 8.72 0.9185 diastolic blood pressure (hgmm) 78.00 10.63 3.21 83.00 13.04 5.83 0.4395 gestational age (weeks) 39.20 1.23 0.39 38.22 2.54 0.85 0.2812 1 minute apgar 9.46 0.52 0.16 8.20 1.1 0.49 0.0213* 5 minute apgar 10.00 0.00 0.00 9.00 1.23 0.55 0.1419 rhohner’s ponderal index 2.69 0.25 0.08 2.85 0.35 0.16 0.1867 bmi of newborn 13.16 0.96 0.32 14.10 1.99 0.89 0.3518 weight (g) 3221 266.1 94.08 3627 363.5 148.40 0.0411* ac (mm) 290.5 34.65 24.50 383.80 29.26 14.63 0.6981 hc (mm) 303.50 9.19 6.50 318.50 16.30 8.15 0.1533 ac/hc 1.04 0.07 0.04 1.28 0.08 0.04 0.1238 bpd (mm) 88.00 4.69 2.34 92.50 3.78 2.18 0.1750 fod (mm) 109.50 5.92 3.42 115.00 9.01 4.50 0.3458 fl (mm) 65.50 6.61 3.30 73.00 4.00 2.31 0.0832 fl/bpd 0.74 0.07 0.04 0.79 0.03 0.02 0.0980 bmi before pregnancy 21.68 3.18 1.20 24.42 5.58 2.79 0.3968 *p<0.05 bmi= body mass index, ac= abdominal circumference, hc= head circumference, bpd= biparietal diameter, fod= frontooccipital diameter, fl= femur length. seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 46 southeastern european medical journal, vol 1, 2017. resistance in placental circulation. in diabetes, the increased blood flow resistance of the umbilical artery is just a late pathognomic sign of ischemic vascular changes in the placenta related to a direct ischemic effect on vascularization. these ischemic-hypoxic alterations can be seen in histopathological samples as syncytial nodes, hypervascularized villi and excessive interstitial calcification (26).we would like to emphasize the importance of the placental-umbilical cord unit in pathologic pregnancies. according to our results, the umbilical cord in the case of a gestational diabetic patient can be described as a thin cord with normal length, hypercoiled or normally coiled rope-like vessels. the incidence of multiple false knots is higher, and they rather tend to have a marginal placental insertion. table 2. the pathomorphological and histopathologic data of the samples. control (n=22) gdm (n=5) mean sd sem mean sd sem p cord cross-sectional area (mm2) 124.00 35.47 11.22 93.75 9.86 4.41 0.0027* cord length (cm) 58.00 9.15 2.89 59.60 7.13 3.19 0.6400 placental weight (g) 463.80 56.77 17.12 535.40 78.53 35.12 0.1083 fetal weight/placental weight 7.38 0.76 0.23 6.29 0.56 0.25 0.0134* placenta thickness (mm) 27.73 4.10 1.24 28.00 4.47 2.00 0.8880 placenta longest diameter (mm) 160.90 13.75 4.15 164.00 5.48 2.45 0.1778 placenta shortest diameter (mm) 139.10 17.00 5.13 158.00 8.37 3.74 0.0071 umbilical coiling index (uci) 1.43 0.77 0.25 2.00 1.00 0.45 0.0891 *p<0.05 figure 1. maximum change in vascular tone after oxytocin dosage. although the mean value for the case group is lower, the difference was not significant; p=0.208 in case of the arteries and p=0.396 in case of the veins. ctrl= control group, gdm= gestational diabetic group. figure 2. vascular tone after oxytocin and desmopressin dosage we considered the spontaneously developing basal tone as 100%.24 seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 47 southeastern european medical journal, vol 1, 2017. on the placental perfusion model, introducing vasopressin to the maternal side (30pg/ml-60 000pg/ml) and increasing the dosage until the vasopressin reaches its maximum concentration on the fetal side, they received 3110 pg/ml as the maximal agent transported through the placenta. this concentration is equivalent with 5.74x10-9m, while our highest concentration was 10-7m. this suggests that treating pregnant women with vasopressin would not influence the fetoplacental circulation. vasopressin or oxytocin concentration still can be increased in vivo by fetal production (27). as described by holcberg g., et.al (2002) oxytocin does have a vasoactive effect on meconium impregnated placentas, but no effect on normal placentas. maternal oxytocin can pass through the placenta and reach the fetal brain, and induce the hyperpolarization of gabaergic neurons in the fetal hippocampus and neocortex during delivery. reduction of gaba-mediated excitation induced by oxytocin has been demonstrated and completely eliminated by atosiban. since hypoxic brain damage is the leading cause of fetal death, the important conclusion is that oxytocin has an inhibitory cortical and hippocampal neuronal effect by which it reduces fetal brain oxygen and nutrient requirements, and therefore it is less sensitive to hypoxia (4). therefore we assume that in cases of placentas without meconium impregnation, oxytocin does not bind to the receptors to elicit vasoconstriction in the placenta, and it can pass to the umbilical cord, where there is no active receptor. thus, it can exert a greater hyperpolarization in the fetal brain. in term pregnancies during delivery, if there is an acute hypoxia, the oxytocin produced by the mother can protect the fetal brain. in contrast, it seems that in cases of the presence of meconium for several hours, it is important to note the increase of vascular tone in the placenta. the macroscopic evaluation of the formalin fixed tissues showed that the volume had reduced, but the ratios between the control and case groups remained the same, so these data correlated with the clinical measurements. in cases of diabetic pregnancies, we found an increased umbilical coiling index (number of 360° turns within 5cm long segment at the placental end). the clinical aspect of the coiling pattern and coiling index is that a thin and hypocoiled cord is more vulnerable to strangulation, striction and other mechanical events. the thin cord that is not wellprotected by wharton’s jelly is prone to striction and mechanical injuries too. there was no significant difference in the case of birth weight and placental weight either, but we did find significant difference in their ratios. the shortest diameter of the gdm placentas was significantly longer than the control ones, while there was no significant difference in the longer diameter or thickness measured at the cord insertion point. therefore, we might observe the diabetic patients’ placentas as more round and less oval shaped, heavier, and exhibiting a pale calcification. we suppose that umbilical vessels have a unique receptor profile to regulate vascular tone though these vessels may injure easily because of mechanical insult. regarding the importance of the umbilical vascular tone in fetal development, the investigation of the exact biochemical mechanisms could be crucial in the prevention of any developmental disturbances since the onset of 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25. peiró c, romacho t, azcutia v, villalobos l, fernández e, bolaños jp, et al. inflammation, glucose, and vascular cell damage: the role of the pentose phosphate pathway. cardiovasc diabetol. 2016;15:82. doi 10.1186/s12933-016-0397-2 26. akarsu s, bagirzade m, omeroglu s, büke b. placental vascularization and apoptosis in type-1 and gestational dm. j matern fetal neonatal med 2016;30(9):1045-1050. doi: 10.1080/14767058.2016.1199676 27. ray jg, boskovic r, knie b, hard m, portnoi g, koren g. in vitro analysis of human transport of desmopressin. clin biochem. 2004;37(1)10-13. seemedj 2021, vol 5, no. 2 anaemia, iron metabolism and covid-19 clinical outcome 9 southeastern european medical journal, 2021; 5(2) review article impact of anaemia and dysregulated iron metabolism on covid-19 clinical outcome – review article 1 matea bogović 1, robert rončević 2, stjepan grga milanković *3 1 department of radiology, national memorial hospital vukovar, vukovar, croatia 2 department of diagnostic and interventional radiology, clinical hospital centre osijek, osijek, croatia 3 department of otorhinolaryngology and head and neck surgery, clinical hospital centre osijek, osijek, croatia *corresponding author: stjepan grga milanković, stjepan.grga.milankovic@gmail.com received: aug 25, 2021; revised version accepted: nov 8, 2021; published: nov 26, 2021 keywords: covid-19, anaemia, iron metabolism, sars-cov-2 abstract coronavirus disease-19 (covid-19) is a disease caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) that can manifest in a wide range of forms, but the most common symptoms are fever, headache, fatigue, respiratory problems, lost sense of smell and taste, sore throat, muscle pain and malaise. in patients with covid-19, the inflammatory response of the organism affects iron homeostasis. severe covid-19 infections may lead to a hyperinflammatory condition, characterised by elevated ferritin levels that correlate with the severity of the clinical course, prolonged intensive care unit (icu) stay, development of acute respiratory distress syndrome and a fatal outcome. as a result of iron metabolism disorders in inflammation, decreased erythropoiesis and reduced biological activity of erythropoietin, the erythrocyte half-life is shortened, leading to anaemia of chronic inflammation. cytokine il-6 plays the most crucial role in regulating iron concentration. it affects iron metabolism by producing hepcidin via stat 3. hepcidin produces regulatory effects on iron by binding with ferroportin, the only known transmembrane iron exporter. anaemia has long been characterised as a significant risk factor contributing to increased mortality and poorer clinical outcomes for various infections. the most severe forms of covid-19 infection result in pneumonia, causing a reduced supply of oxygen to the circulation, ultimately leading to ischemia of vital organs. anaemia of chronic disease is more common in covid-19 positive patients and is associated with a poorer clinical outcome. a higher ferritin/transferrin ratio indicates an advanced inflammatory condition and may be a predictive factor of icu admission. (bogović m, rončević r, milanković sg. impact of anaemia and dysregulated iron metabolism on covid-19 clinical outcome – review article. seemedj 2021; 5(2); 9-17) seemedj 2021, vol 5, no. 2 anaemia, iron metabolism and covid-19 clinical outcome 10 southeastern european medical journal, 2021; 5(2) introduction in the hubei province of the people’s republic of china, in december 2019, a new type of virus emerged – severe acute respiratory syndrome coronavirus 2 (sars-cov-2). the world faced a new global issue, the pandemic of the coronavirus disease 2019 (covid-19). currently, there have been over 200,000,000 coronavirus cases recorded and most patients have presented with mild clinical symptoms (with over 180,000,000 patients recovered). however, 4,000,000 deaths from covid-19 have been recorded up to this moment (1). this pandemic has affected many aspects of our everyday life, from health to economy. in order to provide optimal treatment to patients, it is vital to identify the risk factors contributing to the development of severe forms of the disease so as to effectively use the limited resources available in the fight against the virus. despite the progress that has been made, such as the development of the vaccine, the scientific community is still trying to understand the impact of the virus and its new variants on the clinical outcomes of patients with covid-19. infection with the sars-cov-2 virus can manifest itself in a wide range of clinical presentations: from asymptomatic vectors and milder forms to more severe ones, which require hospital care and respirators, and finally those ending in death. the infection affects various systems and the clinical presentation differs from patient to patient, but the most common symptoms include fever, headache, fatigue, respiratory problems, lost sense of smell and taste, sore throat, muscle pain and malaise (2). a combination of symptoms, medical history information, epidemiological data, polymerase chain reaction (pcr) testing, serological testing, laboratory findings, chest x-ray or computed tomography (ct) findings results in a clinical diagnosis (3). many agents are used in the treatment of covid-19: antiviral drugs, corticosteroids, various antibiotics, antiinflammatory drugs or immunomodulators. over time, the scientific community has discovered alternative forms of treatment and prevention of the disease. at the moment, the best form of protection against infection and the development of more severe forms of the disease is vaccination, which is the most effective weapon in the fight against viruses. in patients with covid-19, the inflammatory response of the organism affects iron homeostasis and leads to a reduction in iron absorption in the intestine, which further reduces the availability of iron for erythropoiesis and haemoglobin production (4). iron is an important redox catalyst in several reactions. due to the fact that a variety of pathogens need iron, one of many defence mechanisms is to limit the availability of iron to infectious agents. for example, iron is mostly found within cells, bound to haemoglobin in erythrocytes. bacteria and viruses have developed mechanisms to steal iron from their hosts (5). the aim of this review is to present the current knowledge from available papers and literature on the complex relationship between iron metabolism, anaemia and covid-19 that affects the clinical course of the disease. regulation of iron metabolism maintenance of iron homeostasis is a complex process involving the regulation of (a) iron in duodenal enterocytes, (b) use in erythroblasts, (c) storage in hepatocytes and (d) macrophage recycling in the spleen. after being reduced with ascorbic acid and duodenal cytochrome b (dcytb) on the apical membrane of enterocytes, iron is absorbed by divalent metal transporter 1 (dmt1) and transferred to the basolateral membrane, where it exits the cells with the help of ferroportin into the circulation, where it binds to transferrin (holo-tf). erythrocytes, the cells that require the most iron, bind holo-tf to the transferrin receptor tfr1. after endocytosis, iron is used in the mitochondria to synthesise haem, which will be incorporated into haemoglobin. if the body absorbs more iron than it needs, it is stored in 6 ferritins, mostly in hepatocytes. the largest seemedj 2021, vol 5, no. 2 anaemia, iron metabolism and covid-19 clinical outcome 11 southeastern european medical journal, 2021; 5(2) source of iron are macrophages, which phagocytose stale erythrocytes and release iron from haem via haem oxygenase 1 (ho1). these processes are regulated by hepcidin, which binds to ferroportin and promotes its internalisation and degradation. in this way, it prevents the absorption of iron and the release of iron stored in hepatocytes and recycled in macrophages. (6) (figure 1). figure 1. maintenance of iron homeostasis inflammation leads to disbalanced iron homeostasis various comorbidities, such as obesity, hypertension, cardiovascular disease, hypercholesterolemia, chronic kidney disease and metabolic syndrome, as well as old age, are associated with higher mortality and poorer treatment outcomes of covid-19 (7). patients suffering from a more severe form of the disease, among other laboratory findings, had leukocytosis with low lymphocyte and platelet count. inflammatory markers, such as c-reactive protein (crp), interleukin 6 (il-6) and ferritin were elevated (8). viral infections, including sars-cov-2 infections, affect the haemoglobin molecule through ace2, cd147, cd26 and other receptors on erythrocytes and/or blood cell precursors. viral endocytosis can cause hemoglobinopathy through the connection between spike proteins and cellular receptors. orf8 protein and surface glycoproteins on the virus can bind to porphyrin, attacking haem on the 1-β chain of haemoglobin. consequently, sars-cov-2 promotes haemolysis and/or creates a haem release complex, forming dysfunctional haemoglobin with a reduced oxygen concentration and co2 transport (9). more severe forms of covid-19 are characterised by an excessive inflammatory response in the form of a cytokine storm, which is characterised by il-6 hyper-expression and hyperferritinemia (figure 2). il-6 also affects iron metabolism by inducing the production of seemedj 2021, vol 5, no. 2 anaemia, iron metabolism and covid-19 clinical outcome 12 southeastern european medical journal, 2021; 5(2) hepcidin via stat 3 (10), which is important in the regulation of iron homeostasis. according to the research published so far, cytokine il-6 plays the most crucial role in regulating iron concentration, at least in humans and animals (11). other cytokines, such as interleukin-1 and activin b, are also involved in regulating iron production, but their role has not been investigated sufficiently (12). hepcidin produces its regulatory effects on iron by binding to ferroportin, the only known transmembrane iron exporter (13). increased hepcidin concentrations inhibit iron absorption in the duodenum, where ferroportin delivers the absorbed iron into the circulation. they also act on macrophages by blocking the release of iron recycled from older erythrocytes into plasma (14). recent research suggests that in higher concentrations, hepcidin can directly block iron exports by occluding ferroportin, a mechanism that may be important in limiting the release of iron from endocytic-deficient cells machines (erythrocytes) or in conditions where endocytosis is slow (15). this mechanism leads to elevated intracellular ferritin. excess intracellular iron reacts with free oxygen radicals, creating oxidative stress. the intracellular iron excess leads to ferroptosis, programmed cell death. excess iron is also thought to cause mitochondrial dysfunction, microbiome diversity and hypercoagulability (16). new evidence suggests that il-6 may have a secondary suppressive effect on erythroid precursors (17). in addition, loss of il-6 and hepcidin results in milder anaemia and faster haemoglobin recovery in a well-established mouse model (18). il-6 knockout animals showed faster bone marrow recovery compared to hepcidin knockout animals. hepcidin downregulates the release of iron into plasma by binding to and functionally lowering ferroportin 1, the sole iron exporter (13,19). persons with high iron levels are at an increased risk of developing various infections. evidence suggests that better control of patients’ iron levels could be a protective factor in the fight against the virus (20). it is still debatable whether an iron metabolism disorder is a result of a physiological response within an infectious disease or it leads to a worse disease outcome. however, recent research has shown that cell damage and inadequate immune response lead to iron disbalance. therefore, we can conclude that iron metabolism disorders significantly contribute to the course of covid-19 (4). impact of impaired iron metabolism on anaemia anaemia has long been characterised as a significant risk factor contributing to increased mortality and poorer clinical outcomes for various infections. it is well known that anaemia exacerbates the severity of respiratory problems, since various diseases and previous studies have shown a high prevalence of anaemia in patients with community-acquired pneumonia (21). although it has not been documented explicitly, the underlying causes and patterns of inflammation, genetic composition and the patient’s pre-disease condition, including iron concentration and erythropoietic capacity, could contribute to each pathophysiological pathway of inflammatory anaemia (22). initially, systemic immune activation leads to significant changes in iron transport, causing iron retention in macrophages and reduced absorption of iron from food. iron sequestration in macrophages is far more critical because 90% of daily iron requirements for haemoglobin (hb) synthesis and erythropoiesis come from recycled iron originating from aged erythrocytes. iron stores in tissues and in the circulation stimulate the expression of hepcidin (11,23) and inflammatory cytokines, hypoxia (12), iron deficiency and ineffective erythropoiesis. inflammation adversely affects erythropoiesis by separating iron from erythroid precursors, thus causing iron-restricted erythropoiesis (24). inflammatory anaemia, a condition involving elevated hepcidin levels, can conceptually be called functional iron deficiency. due to this ironrestricted erythropoiesis, erythroferrone (erfe) increases and is likely to provide some counterregulation of elevated hepcidin. erfe knockout mice treated with heat-killed brucella abortus, a well-accepted model of inflammatory anaemia, showed delayed haemoglobin recovery because of inadequate hepcidin suppression seemedj 2021, vol 5, no. 2 anaemia, iron metabolism and covid-19 clinical outcome 13 southeastern european medical journal, 2021; 5(2) (25). thus, suppressed erythropoiesis in inflammatory anaemia may allow a further increase in hepcidin expression. most covid-19 infections have a milder clinical course and up to 20% of patients require hospital care, most often because of pneumonia, with admission to the icu and a need for mechanical ventilation (26,27). the most severe forms of covid-19 infection result in pneumonia, which leads to diffuse alveolar damage and gas exchange disorders (28). as a result, arterial oxygenation becomes impaired. oxygen saturation depends on the concentration of haemoglobin, which is why reduced haemoglobin levels cause a decrease in the oxygen transfer capacity and oxygen saturation of arterial blood (29). anaemia, as a separate condition, can cause ischemia of vital organs (30). the incidence of anaemia in patients hospitalised in the icu is about 95% (31), while haemoglobin levels are lower in covid-19 positive patients admitted to the icu compared to hospitalised covid-19 positive patients with a milder clinical presentation (32). increased levels of ferritin and hepcidin may be a predictive factor of a poorer clinical outcome. severe covid-19 infections are characterised by a hyper inflammatory condition, . including elevated ferritin levels that correlate with the severity of the clinical presentation, prolonged icu stay, development of acute respiratory distress syndrome and a fatal outcome. as a result of iron metabolism disorders in inflammation, decreased erythropoiesis and reduced biological activity of erythropoietin, the erythrocyte half-life is shortened, leading to anaemia of chronic inflammation (33). a clinical study conducted in china has found that patients with a severe clinical course of covid19 infection had higher levels of hepcidin and ferritin compared to patients with a milder clinical course. it was concluded that hepcidin and ferritin could be predictive factors of the clinical outcome of coronavirus infection (34). also, an elevated ferritin/transferrin ratio is a predictive factor of a worsening clinical condition and longer stay in the icu (31) (figure 2.).. figure 2. increased levels of ferritin and hepcidin may be a predictive factor of a poorer clinical outcome seemedj 2021, vol 5, no. 2 anaemia, iron metabolism and covid-19 clinical outcome 14 southeastern european medical journal, 2021; 5(2) a review of the literature, including prospective studies monitoring the impact of anaemia on the clinical outcome of covid-19 infections, shows that, of the total number of patients hospitalised due to covid-19 infection, the proportion of patients with anaemia ranges between 25% and 65% (33,35,36). studying the difference in the clinical outcome of covid-19 infection in patients with and without anaemia, tao, z. et al. found that patients suffering from anaemia were older and more often had chronic kidney disease. the chinese scientists classified patients with anaemia into three groups according to haemoglobin levels. patients with severe anaemia (hb < 80 g/l) had a higher incidence of dyspnoea and lower levels of o2 partial pressure and o2 saturation compared to patients with mild anaemia (hb between 110 and 119 g/l for women and hb between 110 and 129 g/l for men) and with moderate anaemia (hb between 80 and 110 g/l) (35). patients with severe anaemia were more likely to have coagulation disorders (elevated d-dimers) and increased inflammatory parameters compared to mild-to-moderate anaemia (35). a prospective study conducted in iran by dinevari et al. showed a high prevalence of covid-19 infected patients with anaemia at admission to the hospital as well as an increased risk of admission to icu, need for mechanical ventilation and mortality rates compared to covid-19 positive patients without anaemia. it should also be emphasised that patients with anaemia were older and had more comorbidities, which also increased the risk of a poorer clinical outcome (36). in a study comparing hospitalised covid-19 positive patients with other patients exhibiting the same symptoms, a higher prevalence of anaemia was found among covid-19 positive patients, mainly due to inflammation with elevated ferritin levels and decreased saturated transferrin levels. despite the higher prevalence, no statistically significant higher mortality was observed in anaemic patients compared to covid-19 positive patients without anaemia (37). elevated iron and ferritin levels in the circulation and in the lungs increase the risk of injury to the lung parenchyma (38). severe forms of covid19 have been associated with disseminated intravascular coagulation and thrombosis, but the mechanism of thrombosis is unknown. patients infected with covid-19 have elevated transferrin levels, which may be associated with a hypercoagulable condition (39). it has long been known that transferrin is not only an iron transporter, but it also inhibits antithrombin and promotes the effect of thrombin and coagulation factor xiia (40). the tendency to develop thrombosis is one of the most dangerous complications of covid-19 infection. in order to protect the lungs from excessive free iron, treatment of covid-19 positive patients with lactoferrin and iron chelators has been proposed to reduce circulating free iron (41). however, no clinical study has been published to date to show the effectiveness of this method. regarding covid-19 positive paediatric patients, no significant number of cases involving patients with anaemia have been reported, nor have they had a significantly worse clinical outcome (42). conclusion in conclusion, anaemia of chronic disease is more common in covid-19 positive patients and it is associated with a poorer clinical outcome. a higher ferritin/transferrin ratio reflects an advanced inflammatory condition and may be a predictive factor of icu admission and the need for mechanical ventilation. given the important role of iron metabolism in covid-19 infection, future studies should evaluate the efficacy of treatment with iron chelators and lactoferrin and their impact on the clinical outcome of these diseases. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare seemedj 2021, vol 5, no. 2 anaemia, iron metabolism and covid-19 clinical outcome 15 southeastern european medical journal, 2021; 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10(8):539. doi: 10.3390/diagnostics10080539 40. tang x, zhang z, fang m, han y, wang g, wang s, xue m, li y, zhang l, wu j, yang b, mwangi j, lu q, du x, lai r. transferrin plays a central role in coagulation balance by interacting with clotting factors. cell research. 2019; 30(2):119-132. doi: 10.1038/s41422-019-0260-6 41. habib h, ibrahim s, zaim a, ibrahim w. the role of iron in the pathogenesis of covid-19 and possible treatment with lactoferrin and other iron chelators. biomedicine pharmacotherapy. 2021; 136:111228. doi: 10.1016/j.biopha.2021.111228 42. kosmeri c, koumpis e, tsabouri s, siomou e, makis a. hematological manifestations of sars‐cov‐ 2 in children. pediatric blood cancer. 2020 dec;67(12):e28745. doi: 10.1002/pbc.28745.. 1 author contribution. acquisition of data: bogović m, rončević r, milanković sg administrative, technical or logistic support: bogović m, rončević r, milanković sg conception and design: bogović m, rončević r, milanković sg critical revision of the article for important intellectual content: bogović m, rončević r, milanković sg drafting of the article: bogović m, rončević r, milanković sg final approval of the article: bogović m, rončević r, milanković sg seemedj 2022, vol 6, no 1 dheas and hypertension 68 southeastern european medical journal, 2022; 6(1) review article dehydroepiandrosterone sulfate and arterial hypertension 1 juraj jug 1*, marina matovinović 2 1 health center zagreb-west, zagreb, croatia 2 university hospital center, zagreb, croatia *corresponding author: juraj jug, juraj2304@gmail.com introduction dehydroepiandrosterone sulfate (dheas) is a steroid hormone created by adding a sulfate group to dehydroepiandrosterone (dhea) in the zona reticularis of the suprarenal gland (90%) and the gonads (10%). dhea is also synthesized in the central nervous system, where its concentration is 6–8 times higher than in the received: jan 6, 2022; revised version accepted: mar 11, 2022; published: apr 27, 2022 keywords: arterial hypertension, dehydroepiandrosterone sulfate, menopause, neuroprotection serum, which is why it is also called a neurosteroid hormone (1). depending on age, dheas can serve as a precursor in the production of other hormones (2). accordingly, in the reproductive age, 40–75% of testosterone is synthesized from dheas. on the other hand, more than 90% of circulating estrone is created from dheas (3). dhea abstract dehydroepiandrosterone sulfate (dheas) is a steroid molecule whose function and mechanism of action in the human body are still inadequately researched. a potential protective function for the cardiovascular system can be explained by activation of nitric oxide production, impact on endothelial and mitochondrial function, and inhibition of proinflammatory cytokine production (il-6 and tnf-α). some research shows the beneficial effects of dhea/dheas on many bodily functions, especially in the cardiovascular and the neurological systems. however, we need to be careful with interpretation of the results because of different criteria used for defining arterial hypertension, the race that was observed, and reproductive status of women, as these factors can change the conclusion. due to a lack of evidence, dheas supplementation is still not recommended. we need multicentric prospective and randomized studies on dheas to examine its potential impact on blood pressure regulation and cardiovascular risk. (jug j, matovinović m. dehydroepiandrosterone sulfate and arterial hypertension. seemedj 2022; 6(1); 68-73) seemedj 2022, vol 6, no 1 dheas and hypertension 69 southeastern european medical journal, 2022; 6(1) concentration in the serum is 10 times higher than cortisol and it shows weak androgenic and estrogenic activity. on the other hand, dheas is a hormonally inert molecule with the greatest activity in the central nervous system; it is 100 times more concentrated in the serum than cortisol. this concentration makes it the steroid with the highest ratio in the serum (4). in addition, its levels significantly depend on gender and age, with the lowest levels found in childhood and the period before puberty, while in puberty males have dheas levels twice as high as females, with the maximum reached in their early 20s. in women, these levels are not dependent on the menstrual cycle. total dheas concentration decreases by 2% annually and after the age of 70, it reaches pre-puberty levels. the half-life of dheas in the serum is around 10 hours. in comparison, dhea has a 20 times shorter half-life than dheas (30 minutes) (5). in recent research, dheas has been shown to have a positive impact on learning and memorizing due to allosteric modification of nmethyl-d-aspartate (nmda) receptors. likewise, dheas can exaggerate neuron excitation through negative allosteric modulation of gamma-aminobutyric acid (gabaa) receptors (6). by activating sigma-1 receptors and producing nerve growth factor (ngf) and brain-derived neurotrophic factor (bdnf), dheas serves a neuroprotective function (7–9). in addition to effects on the central nervous system, dheas has other pleiotropic effects, including modulation of mitochondrial and endothelial function, as well as bone metabolism (10). one minute of stimulation of bovine aortic endothelial cells (100 nm for 5 min) with dhea (abolished responses with nitro-larginine methyl ester (l-name) or phosphatidylinositol (pi) 3 – kinase inhibitor) increased nitric oxide production, stimulated endothelial cell growth, decreased the growth of smooth muscles in blood vessels and the levels of plasminogen activator inhibitor type 1 (pai-1), and inhibited leukocyte adhesion (11–13). likewise, dhea can inhibit the production of proinflammatory cytokines such as interleukin-6 (il-6) and tumor necrosis factor α (tnf-α) (11, 14). the release of no is independent of intracellular calcium mobilization but depends on g protein tyrosine and mitogen-activated protein (map) kinases (15). this mechanism of action could be the key to the cardioprotective effect proposed for dhea. dhea and dheas levels are often elevated in polycystic ovary syndrome (pcos), congenital adrenal hyperplasia (cah), cushing’s syndrome, and in the presence of hormonally active tumors (16). history of dheas research and cardiovascular risk in 1975, by examining dhea and dheas concentrations by compartmentalization in urine and blood, feher et al (17) suggested that a significant uptake of dhea by adipose tissue occurs, causing lower sulfation into dheas and its faster metabolism in obese female patients. they also connected low dheas concentration with increased insulin resistance in three obese individuals. later, in 1987, rotter et al (18) examined the distribution of dheas levels in 178 individuals from 26 families and correlated low dheas levels with low testosterone levels. barret-connor et al (19) examined the connection between low dheas levels and 12year mortality in 242 men aged 50 to 79. dheas levels below 3.8 μmol/l showed a relative risk of 1.5 for death from any cause (without statistical significance), 3.3 (p < 0.05) for death from cardiovascular (cv) disease, and 3.2 (p < 0.05) for death from ischemic heart disease in males. there was still a lot to learn about dheas in the coming years, and barret-connor et al (20) expanded their cohort to 1,029 men and 942 women in california, usa, in 1995, linking low dheas levels with coronary ischemic disease in postmenopausal women, but without increased cv mortality. the next significant research was conducted in 1997, when barna et al (21) found a significant correlation between serum dheas concentration and blood pressure (including the dipping profile), from which they concluded that seemedj 2022, vol 6, no 1 dheas and hypertension 70 southeastern european medical journal, 2022; 6(1) lower dheas levels are associated with higher blood pressure. this research was conducted on 387 white subjects (86 normotensive and 301 hypertensive), suggesting a positive effect of dheas supplementation on cv risk. two years later, in 1999, schunkert et al (22) made the opposite conclusion in their study conducted on 646 subjects, associating higher dheas levels with higher blood pressure and higher levels of aldosterone. the debate was resolved in 2004 with the discovery by liu et al (15), who found that dheas increases the activity of nitric oxide synthase (enos) mediated by tyrosine and map kinases in both bovine aortic endothelial cells and human umbilical vein endothelial cells. further research on dheas was rarely conducted until 2010, when the wise study (women’s ischemia syndrome evaluation), conducted on 270 postmenopausal women, showed that lower dheas levels are linked to higher cv mortality in postmenopausal women who underwent coronarography due to myocardial ischemia (23). furthermore, in 2019, de paiva lemos et al (24) found a correlation between dheas and lower heart rate variability in old age, but this causality can hardly be confirmed since heart rate variability decreases physiologically with aging, much like dheas serum concentration. the other problem with this study was that the sample consisted of only 45 men, divided into three age groups. however, today we assume that lower heart rate variability is linked to a 32–45% higher risk of cv events (25). possible reasons why the aforementioned researchers came to different conclusions about dheas and its impact on blood pressure may lie in the choice of methods and patients. to date, only three studies have examined the correlation between dheas and blood pressure levels. differences in the methodology used in these studies are shown in table 1. table 1. methodology of the most prominent studies about dheas and arterial hypertension authors and year correlation between dheas and ah correlation with nocturnal indices ah criteria race (country) barna et al, 1997 (21) negative positive > 130/85 mmhg white (hungary) schunkert et al, 1999 (22) positive / > 160/95 mmhg white (germany) jimenez et al, 2019 (26) nonexistent / > 130/80 mmhg latino (puerto rico) ah = arterial hypertension . dheas supplementation due to the previously mentioned benefits for the cv system and the brain, scientists have been discussing dheas supplementation in postmenopausal women for many years. yet, meta-analyses conducted by boxer et al. (27) in 2010 and by wang et al. (28) in 2020 did not show any clear benefits of dheas supplementation in postmenopausal women. longer prospective cohort studies are needed to accurately demonstrate the benefits of this kind of supplementation in women. medication and dheas higher dheas serum levels were observed in hypertensive patients treated with betablockers and calcium channel blockers (16). on the other hand, lower dheas levels were observed in patients treated with ace inhibitors (29). seemedj 2022, vol 6, no 1 dheas and hypertension 71 southeastern european medical journal, 2022; 6(1) lifestyle changes and dheas despite the clear evidence of effects of a healthy diet and weight loss on blood pressure, the impact of lifestyle changes on dheas levels remains unknown. the exception is smoking, since nicotine can stimulate the production of corticotropin-releasing hormone (crh) and vasopressin, which stimulate dheas synthesis (30). likewise, smoking can decrease dheas clearance, as well as its binding to serum proteins, which also increases its serum concentration (31). diet rich in pectin can likewise increase dheas levels in the serum, but excessive protein intake may reduce dhea levels (32, 33). an increase of dhea levels immediately after exercise was found in both genders, but dheas levels increased only in women (34). a very small number of studies have examined the impact of exercise on these hormones, with many methodological differences, which complicates their comparison.cardiovascular and renal disease in non-diabetic individuals with arterial hypertension remains a question to be answered. conclusion although there is clear evidence that dheas can stimulate no production in blood vessels, the conclusions of relatively small and methodologically different studies about its impact on blood pressure are still contradictory. however, some studies have shown increased cv mortality in subjects with low dheas levels, but its supplementation is still not recommended. there is a need for larger prospective multicentric studies to be conducted in order to clarify the connection between dheas and arterial hypertension. acknowledgement. none. disclosure funding. no specific funding was received for this study competing interests. none to declare. references 1. baulieu ee, robel p. dehydroepiandrosterone (dhea) and dehydroepiandrosterone sulfate (dheas) as neuroactive neurosteroids. proc natl acad sci u s a 1998; 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74(1):139-43. doi: 10.1210/jcem.74.1.1530789. 31. friedman aj, ravnikar va, barbieri rl. serum steroid hormone profiles in postmenopausal smokers and nonsmokers. fertil steril. 1987; 47(3):398-401. 32. remer t, pietrzik k, manz f. the shortterm effect of dietary pectin on plasma levels and renal excretion of dehydroepiandrosteron sulfate. z ernahrungswiss. 1996; 35:32-38. 33. trichopoulou a, bamia c, kalapothaki v, spanos e, naska a, trichopoulos d. dehydroepiandrosterone relations to dietary and lifestyle variables in a general population sample. ann nutr metab. 2003; 47:158-164. doi: 10.1159/000070039 34. heaney jl, carroll d. dhea, dhea-s and cortisol responses to acute exercise in older adults in relation to exercise training status and sex. age. 2013; 35:395-405. doi: 10.1007/s11357011-9345-y author contribution. acquisition of data: jj administrative, technical or logistic support: jj, mm analysis and interpretation of data: jj, mm conception and design: jj, mm critical revision of the article for important intellectual content: jj, mm drafting of the article: jj, mm final approval of the article: jj, mm seemedj 2022, vol 6, no 1 corneal edema recovery 113 southeastern european medical journal, 2022; 6(1) original article postoperative corneal edema after phacoemulsification 1 martina liovic milec 1*, sandra sekelj 1, slavica konjevic-pernar 2 1 department of ophthalmology, general hospital “dr. josip benčević”, slavonski brod, croatia 2 department of ophthalmology, general hospital “nova gradiška”, nova gradiška, croatia *corresponding author: martina liovic milec, martinaliovic@yahoo.com received: feb 8, 2022; revised version accepted: mar 17, 2022; published: apr 27, 2022 keywords: cataract, corneal edema, phacoemulsification, visual acuity abstract aim: to determine the effect of nuclear opalescence (no) on intraoperative parameters during phacoemulsification using the whitestar signature® pro and to show the impact of preoperative and intraoperative parameters on postoperative corneal edema. methods: this prospective study included 267 patients selected to undergo phacoemulsification using the whitestar signature® pro system at the department of ophthalmology of the general hospital “dr josip benčević”, slavonski brod, croatia. no was graded using the lens opacities classification system iii. preoperative parameters were age, sex, no and preoperative central corneal thickness. intraoperative parameters of phacoemulsification included in the study were ultrasound time (ust), phaco time using ellips fx technology (efx) and average phaco power (avg). patients were followed up on postoperative days 1 and 7 and after two months. the state of the cornea was noted in each follow-up. results: there was a statistically significant increase of intraoperative parameters with no. postoperative corneal edema depended on all measured intraoperative parameters (ust, efx and avg, all p < .001), patient’s age (p < .05) and no (p < .001) on postoperative day 1, while on postoperative day 7, it depended on ust (p = .011) efx (p = .012) and no (p < .05). conclusion: older patients, higher grade of no and amount of energy consumed during phacoemulsification using the whitestar signature® pro are predictive factors for severity of transient corneal edema. we found this information important for better preoperative planning of phacoemulsification, as well as for better postoperative results. (liovic milec m, sekelj s, konjevic-pernar s. postoperative corneal edema after phacoemulsification. seemedj 2022; 6(1); 113-120) seemedj 2022, vol 6, no 1 corneal edema recovery 114 southeastern european medical journal, 2022; 6(1) introduction continuous advances in phacoemulsification have made safer and more efficient cataract surgery possible. there is a need for objective and transparent research in order to observe the impact of new technology on surgery outcomes, patient safety and satisfaction. the most frequent complication after phacoemulsification is corneal edema (1). the main factor that determines the function of the cornea as clear and transparent tissue is a metabolically active monolayer, corneal endothelium (2). corneal endothelial cell loss after phacoemulsification leads to corneal edema (3), which may be due to perioperative and intraoperative risk factors. intraoperative risk factors which can lead to acute corneal edema after phacoemulsification are endothelial injury by consumed ultrasound energy, descemet’s membrane detachment, toxic anterior segment syndrome (tass) and iol instability with endothelial touch (1). around 2010, abbott medical optics (amo) introduced the new ellips fx system, which combined both longitudinal and elliptical motion into a single handpiece to improve phaco efficiency. the whitestar signature® pro was introduced by amo in 2015 and has demonstrated improved nucleus followability, cutting efficiency and proactive intraocular pressure management with automatic occlusion sensing (4, 5). in a previously published article by sekelj et al., corneal edema recovery was compared between patients with type 2 diabetes mellitus and patients without type 2 diabetes (6). after that, we wanted to analyze the use of the new phaco technology in our hospital on postoperative corneal edema recovery. the aims of this study were to determine the effect of nuclear opalescence (no) on intraoperative parameters during phacoemulsification using the whitestar signature® pro with the ellips fx handpiece and to determine the impact of preoperative and intraoperative parameters on postoperative corneal edema recovery. patients and methods this prospective study included 267 patients selected to undergo phacoemulsification using the whitestar signature® pro system. the phacoemulsification surgery was performed on all patients by a single experienced surgeon during the study period of 7 months at the department of ophthalmology in our hospital. the study was performed in accordance with the principles of the helsinki declaration. informed consent was obtained from all study participants. out of 267 patients who were included in the study, 266 patients came to checkup on the first postoperative day, 265 patients came to checkup on the seventh postoperative day and 205 patients came to checkup after 2 months. other study participants were under care in other hospitals. we performed preoperative examination and used exclusion criteria (previous ocular infection or ocular surgery and fuchs’ endothelial corneal dystrophy) on all patients using the previously described methods.6 three independent observers graded cataracts using a slit lamp and the findings were compared based on the lens opacities classification system iii (locs iii).7 eyes with no grades between 1 and 6 were included in the study and were subdivided into mild (no1, no2), moderate (no3, no4) and hard (no5, no6) cataracts. phacoemulsification was performed with a 2.75 mm clear corneal incision. sodium hyaluronate (healon gv) was injected in the anterior chamber. one side port was created using a 30 degree knife, followed by continuous curvilinear capsulorhexis and hydrodissection. phacoemulsification was performed using the whitestar signature® pro system, using the phaco chop technique with the ellips fx handpiece (amo, inc.). coaxial irrigation/aspiration of the remaining cortical lens material was performed. intraocular lens was implanted in the capsular bag after injecting hydroxypropyl methylcellulose 2.0% (celoftal). the “rock ‘n’ roll” (rnr) technique was used for removing ophthalmic viscosurgical devices at the end of the surgery. the clear seemedj 2022, vol 6, no 1 corneal edema recovery 115 southeastern european medical journal, 2022; 6(1) corneal incision wound was hydrated and prophylactic intracameral injection of cefuroxime or vancomycin was given. postoperative treatment included the use of dexamethasone/neomycin/polymyxin b (maxitrol) drops and bromfenac drops in all type 2 diabetic patients based on our previously described protocol (6). ultrasound time (ust), phaco time using ellips fx technology (efx) and average phaco power (avg) were intraoperative parameters for phacoemulsification. the efx is a parameter of effective phaco time (ept), with the specific coefficient for transversal movement expressed in seconds. patients were followed up on postoperative days 1 and 7 and after 2 months. two independent observers graded postoperative corneal edema recovery using three grading scales: clear cornea, focal corneal edema and diffusive corneal edema, as previously described (6). statistical analysis was performed using spss software (version 24, spss inc, ibm corporation, chicago, usa). the shapiro–wilk test was used to check if a continuous variable follows a normal distribution. significance was tested using the wilcoxon signed-rank test, the kruskal–wallis h test, the friedman test and the chi-squared test. a p value of less than 0.05 was considered statistically significant. results the study was conducted in order to determine the effect of no on intraoperative parameters (ust, efx, avg) during phacoemulsification using the whitestar signature® pro with the ellips fx handpiece. the other reason was to establish the impact of preoperative (age, sex, no, precct) and intraoperative parameters on postoperative corneal edema recovery. there were 267 patients included in the study; 164 were females (61%) and 103 were males (39%). the average age of the study population was 73.0 ± 8.76 years. most patients had moderate cataract (67%). mean precct was 556 (range 445–697). intraoperative parameters are shown in table 1. ust, efx and avg were statistically significantly different based on estimated nuclear hardness (kruskal–wallis h test, all p < .001) (table 1); all intraoperative parameters increased significantly with no. ust, efx and avg were statistically significantly different between mild and moderate cataract (wilcoxon signed-rank test, all p < .001, post hoc test), moderate and hard cataract (wilcoxon signed-rank test, all p < .001, post hoc test), as well as mild and hard cataract (wilcoxon signedrank test, all p < .001, post hoc test). significantly higher mean ust, efx and avg were observed in hard cataracts, indicating a greater amount of energy during phacoemulsification. table 1. ultrasound time (ust), elliptical motion energy (efx) and average phaco power (avg) values for various grades of cataract (n = 267) mild cataract moderate cataract hard cataract kruskal– wallis h test (average ± sd) (average ± sd) (average ± sd) ust (seconds) 5.6 ± 8.25 37 ± 20.25 87.2 ± 33.3 p < .001 efx 1.3 ± 2.25 12.7 ± 9.33 38.7 ± 17.45 p < .001 avg (%) 1.8 ± 2.25 5.8 ± 2.32 8.2 ± 1.54 p < .001 seemedj 2022, vol 6, no 1 corneal edema recovery 116 southeastern european medical journal, 2022; 6(1) table 2. intraoperative parameters (ust, efx, avg) in connection with postoperative corneal edema on postoperative day 1 (n = 266) + (n = 173) ++ (n = 68) +++ (n = 25) kruskal– wallis h test average ± sd average ± sd average ± sd ust (sec) 30.8 ± 28.22 49.2 ± 30.85 68.4 ± 32.88 p < .001 efx 11.0 ± 12.59 19.7 ± 17.14 25.4 ± 15.71 p < .001 avg (%) 4.8 ± 2.94 (5) 6.4 ± 2.74 7.0 ± 1.92 p < .001 + clear cornea, ++ focal corneal edema, +++ diffusive corneal edema another area of interest was postoperative corneal edema, as well as determining the impact of preoperative and intraoperative parameters on postoperative corneal edema recovery. in our study, postoperative corneal edema depended on all measured intraoperative parameters on postoperative day 1 (all kruskal–wallis h test, p < .001) (table 2), while on postoperative day 7, it depended on ust and efx (wilcoxon signed-rank test, p = .011, p = .012), with no correlation with avg (wilcoxon signed-rank test, p = .179) (table 3). table 3. intraoperative parameters (ust, efx, avg) in connection with postoperative corneal edema on postoperative day 7 (n = 265) + (n = 255) ++ (n = 10) wilcoxon signed-rank test average ± sd average ± sd ust (seconds) 37.4 ± 30.42 72.2 ± 43.61 p = .011 efx 13.9 ± 14.54 27.9 ± 19.72 p = .012 avg (%) 5.4 ± 2.94 6.6 ± 2.41 p = .179 + clear cornea, ++ focal corneal edema in the study, we observed a statistically significant reduction in postoperative corneal edema (friedman test = 126.4, p < .001). there were statistically significant differences between postoperative days 1 and 7 (wilcoxon signed-rank test = -8.514, p < .001), as well as between postoperative day 7 and two months after the operation (wilcoxon signed-rank test = -2.828, p < .01). in a previously published article, we compared corneal edema recovery and visual acuity between patients with type 2 diabetes mellitus and patients without type 2 diabetes (6). furthermore, in this study, we analyzed the impact of preoperative parameters on postoperative corneal edema. corneal edema depended on the patient’s age and no on postoperative day 1 (table 4), while on postoperative day 7, it depended only on no (table 5). in this study period, there were no cases of bullous keratopathy. seemedj 2022, vol 6, no 1 corneal edema recovery 117 southeastern european medical journal, 2022; 6(1) table 4. preoperative parameters (sex, patient’s age, no, precct) in connection with postoperative corneal edema on postoperative day 1 (n = 266) + (n = 173) ++ (n = 68) + + + (n = 25) % average ± sd % average ± sd % average ± sd sex female 64.0 28.7 7.3 p = .162 male 66.7 20.6 12.7 * patient’s age 72.2 ± 8.13 73.6 ± 9.89 76.8 ± 8.75 p < .05 ** no mild 91.5 8.5 0 moderate 63.7 27.9 8.4 p < .001 hard 40.0 35.0 25.0 * precct 555.9 ± 37.37 558.1 ± 41.09 552.9 ± 30.52 p = .791** + clear cornea, ++ focal corneal edema, +++ diffusive corneal edema, χ2*, kruskal–wallis h test** table 5. preoperative parameters (sex, patient’s age, no, precct) in connection with postoperative corneal edema on postoperative day 7 (n = 265) + (n = 255) ++ (n = 10) % average ± sd % average ± sd sex female 96.9 3.1 p = .462 male 95.1 4.9 * patient’s age 73.1 ± 8.7 70.7 ± 10.39 p = .58 ** no mild 100 0 moderate 96.6 3.4 p < .05 hard 89.7 10.3 * precct 555.7 ± 38.03 568.9 ± 28.24 p = .2** + clear cornea, ++ focal corneal edema, +++ diffusive corneal edema, χ2*, ** wilcoxon signed-rank test discussion transient postoperative corneal edema leads to dissatisfied patients, which is why there is a need to evaluate preoperative and intraoperative risk factors for endothelial pump failure in the use of modern phaco technology. in the present study, 67% of the patients had moderate cataracts, 17.6% of the patients had mild cataracts and 15% of the patients had hard cataracts. significantly higher mean ust, efx and avg (mean 87.2, 38.7 and 8.2, respectively) were observed in hard cataracts than in mild cataracts (mean 5.6, 1.3 and 1.8, respectively) or moderate cataracts (mean 37, 12.7 and 5.8, respectively). this is consistent with what has been found in previous studies, showing that the average phacoemulsification time increases due to increasing grades of lens hardness (1, 2, 8-15). in conclusion, we can assume that cataract density is a predictive factor for the amount of energy used during phacoemulsification. seemedj 2022, vol 6, no 1 corneal edema recovery 118 southeastern european medical journal, 2022; 6(1) cataract surgery is one of the most common surgical procedures in the world, with a positive effect on the patients’ quality of life. since postoperative corneal edema leads to dissatisfied patients, another area of interest was postoperative corneal edema and the time needed for corneas to become clear after phacoemulsification using the whitestar signature® pro with the ellips fx handpiece. postoperative corneal edema was present in 93 eyes (35%) on postoperative day 1, and most of the patients had focal corneal edema (25.6%), which decreased to 3.8% on postoperative day 7. a similar pattern of results was observed by kausar et al., who showed that postoperative corneal edema was present in 44% of the patients and that most of the patients had focal corneal edema (24.7%) (8). based on the results of our study, predictive factors for assessment of postoperative corneal edema are no and the patient’s age. this is consistent with what has been found in a previous study (8). this observation might be explained by the theory that significant factors for corneal clarity are the number and condition of endothelial cells. other than quantity, the quality of endothelial cells is also an important factor for corneal clarity (2). gradual corneal endothelial cell loss occurs with increasing age and studies estimate the cell loss rate at 0.3% to 0.5% per year (3, 16). with regard to the corneal endothelial barrier and pump function, our study suggests that there is a higher risk of transient postoperative corneal edema in older patients. the limitation of the present study is subjective evaluation of postoperative corneal edema using slit lamp biomicroscopy due to a lack of resources. one of the intraoperative risk factors which can lead to transient corneal edema after phacoemulsification is endothelial injury caused by consumed ultrasound energy (1). in order to reduce the amount of energy used during phacoemulsification, we used the phaco chop technique, since it has been shown in previous studies that less ultrasound energy is needed when using phaco chop in comparison with other techniques (8, 17,18). in our study, we used the ellips fx handpiece with a combined longitudinal and transversal movement of the tip, which demonstrates safe performance in comparison with the standard longitudinal phaco handpiece, especially when dealing with hard cataracts (5). likewise, in our study, only 3.8% of the patients had focal corneal edema on postoperative day 7, and all the patients had clear cornea two months after the operation, which confirmed the surgical efficiency of the ellips fx handpiece. the results of this study showed that corneal edema depend on measured intraoperative parameters (ust, efx and avg), showing that consumed energy is a predictive factor for the severity of corneal edema, as shown in the study by kausar et al (8). on the other hand, the study by tsaosus and al. showed that ultrasound energy is not the determining factor for corneal edema (2). this study aimed to analyze the use of advanced phaco technology with the whitestar signature® pro system with the ellips fx handpiece, based on cataract density and its effect on postoperative corneal edema. to conclude, using advanced technology makes every surgeon’s life easier. the whitestar signature® pro with the ellips fx handpiece showed effective surgical performance with good corneal edema recovery in our study population. despite continuous advances in cataract surgery, there is still a higher risk of transient postoperative corneal edema in harder cataracts, which require a greater amount of energy use during phacoemulsification, especially in the older population. further research is needed to better understand the risks associated with postoperative corneal edema. acknowledgement. none disclosure funding. none. competing interests. none to declare. seemedj 2022, vol 6, no 1 corneal edema recovery 119 southeastern european medical journal, 2022; 6(1) references 1. tsaousis kt, panagiotou dz, kostopoulou e, vlatsios v, stampouli d. corneal oedema after phacoemulsification in the early postoperative period: a qualitative comparative case-control study between diabetics and nondiabetics. ann med surg (lond). 2015; 5:67-71. doi: 10.1016/j.amsu.2015.12.047 2. sharma n, singhal d, nair sp, sahay p, sreeshankar ss, maharana pk. corneal edema after phacoemulsification. indian j ophthalmol. 2017; 65(12):1381-1389. doi: 10.4103/ijo.ijo_871_17 3. lundberg b, jonsson m, behndig a. postoperative corneal swelling correlates strongly to corneal endothelial cell loss after phacoemulsification cataract surgery. am j ophthamol. 2005; 139(6):1035-41. doi:10.1016/j.ajo.2004.12.080 4. kim yn, lee ja, kim jy, kim mj, tchah hw. clinical effects of an improved pump reaction rate and automatic occlusion sensing system in phacoemulsification. j korean ophthalmol soc 2018; 59(11):1017-1023. doi:10.3341/jkos.2018.59.11.1017 5. tognetto d, d’aloisio r, cecchini p, di nicola m, di martino g. comparative clinical study of whitestar signature phacoemulsification system with standard and ellips fx handpieces. int ophthalmol. 2017; 38(4):1697–1702. doi: 10.1007/s10792-017-06495 6. sekelj s, liovic m, konjevic-pernar s, sekelj a, farena s. corneal edema recovery after phacoemulsification in type 2 diabetic versus non-diabetic patients. clinical diabetology. 2021; 10(1):144-148. doi: 10.5603/dk.a2021.0003 7. chylack ltj, wolfe jk, singer dm, leskemc, bullimore ma, bailey il, friend j, mccarthy d, wu s. the lens opacities classification system iii. arch ophthalmol. 1993; 111:831–836. doi:10.1001/archopht.1993.01090060119035 8. kausar a, farooq s, akhter w, akhtar n. transient corneal edema after phacoemulsification. j coll physicians surg pak. 2015; 25(7):505-509. doi: 07.2015/jcpsp.505509 9. ojha t. study of nuclear hardness and phaco time in phacoemulsification. int j sci stud. 2017; 5(9):44-47. doi: 10.17354/ijss/2017/553 10. al-khateeb g, shajari m, vunnava k, petermann k, kohnen t. impact of lens densitometry on phacoemulsification parameters and usage of ultrasound energy in femtosecond laser-assisted lens surgery. can j ophthalmol. 2017; 52(4):331-337. doi: 10.1016/j.jcjo.2017.01.004 11. bečnić g, zorić-geber m, šarić d, čorak m, mandić z. locs iii and phacoemulsification. coll. antropol. 2005; 1:91–94. 12. ramamurthy lb, venugopal kc, acharya p, manipur sr. comparison of effective phaco time and ultrasound time among 2.8 mm and 2.2 mm phacoemulsification in various grades of cataract. j clin ophthalmol res. 2018; 6:12-4. doi: 10.4103/jcor.jcor_68_16 13. al-osaily am, al-jindan my. intracorrelations between cataract density based on scheimpflug image, phacodynamics, surgery duration, and endothelial cell loss after phacoemulsification. saudi j ophthalmol. 2018; 32(3):188–193. doi: 10.1016/j.sjopt.2018.04.004 14. lim sa, hwang j, hwang ky, chung sh. objective assessment of nuclear cataract: comparison of double-pass and scheimpflug systems. j cataract refract surg. 2014; 40:716– 721. doi: 10.1016/j.jcrs.2013.10.032 15. demircan s, atas m, koksal m, pangal e, yuvaci i, goktas a. relationship between lens density measurements by pentacam scheimpflug imaging and torsional phacoemulsification parameters. international eye science. 2014; 14:1739-1743. doi:10.3980/j.issn.1672-5123.2014.10.0 16. woodward ma, edelhauser hf. corneal endothelium after refractive surgery. j cataract refract surg. 2011; 37:767–777. doi: 10.1016/j.jcrs.2011.01.012 seemedj 2022, vol 6, no 1 corneal edema recovery 120 southeastern european medical journal, 2022; 6(1) 17. storr-paulsen a, norregaard jc, ahmed s, storr-paulsen t, hyldebrandt pedersen t. endothelial cell damage after cataract surgery: divide-and-conquer versus phaco-chop technique. j cataract refract surg. 2008; 34:9961000. doi: 10.1016/j.jcrs.2008.02.013 18. debry p, olson rj, crandall as. comparison of energy required for phaco-chop and divide and conquer phacoemulsification. j cataract refract surg. 1998; 24(5):689-692. doi: 10.1016/s0886-3350(98)80267-8. author contribution. milec ml and sekelj s were responsible for the conceptualization and design of the study. milec ml, sekelj s and pernar sk collected the data and were responsible for data validation. milec ml and sekelj s performed the statistical analysis and supervised the study. milec ml wrote the manuscript. sekelj s reviewed and edited the manuscript. all authors have read and agreed on the published version of the manuscript. seemedj 2022, vol 6, no 1 micrornas and hypertension 53 southeastern european medical journal, 2022; 6(1) review article micrornas and hypertension 1 nikolina kolobarić 1,2, ines drenjančević 1,2* 1 department of physiology and immunology, faculty of medicine osijek, josip juraj strossmayer university of osijek, croatia 2 scientific center of excellence for personalized health care, josip juraj strossmayer university of osijek, croatia *corresponding author: ines drenjančević, ines.drenjancevic@mefos.hr received: mar 11, 2022; revised version accepted: mar 28, 2022; published: apr 27, 2022 keywords: blood pressure, hypertension, micrornas, sodium chloride abstract micrornas (mirnas) are non-coding, highly conserved rnas found in all biological fluids, that are emerging as master regulators of gene expression, consequently impacting a variety of biological processes in both healthy and diseased environments. there are still certain limitations regarding analysis of circulating mirnas, specifically concerning standardisation and accuracy of obtained data. however, there is an indisputable therapeutic and diagnostic potential, confirmed by recent research. hypertension, as one of the leading causes of death in modern world, has been in the focus of scientific society for several decades now. so, it is of outmost importance to investigate and pinpoint appropriate mirnas for early indication and diagnosis of hypertension in general population. more in vivo and clinical research is necessary in animal and human models in order to exploit the full potential of this novel technology. (kolobarić n, drenjančević i. micrornas and hypertension. seemedj 2022; 6(1); 53-67) seemedj 2022, vol 6, no 1 micrornas and hypertension 54 southeastern european medical journal, 2022; 6(1) introduction micrornas (mirnas) represent a class of short length (20-24 nucleotides), highly conserved, non-coding rnas that have a role in regulation of gene expression through inhibition of transcription, translation or degradation of target genes, depending on their origin (1,2). mirnas can be categorized as tissue-derived mirnas (tmirnas) and freely circulating mirnas (cmirnas), with t-mirnas being mainly associated with hypertension and cardiac function (more evidence needed), while the latter are described as a potential specific biomarkers for early disease detection, with their abnormal expression being largely associated with diseases in humans (1,3). c-mirnas or extracellular mirnas can be found in all biological fluids, mainly carried in vesicles exosomes, such as serum, plasma, saliva, urine, while serving a hormone-like purpose in processes of signalisation, mediation and regulation of a variety of biological and cellular activities, physiological responses and pathological conditions (3–5). even though the sampling is minimally invasive and there is a potentially high reproducibility of results, analysis of mirnas still represents a challenge due to low concentrations of target compounds, lack of standardized methodology and eventual accuracy obtained regarding their in vivo role and therapeutic properties (1,6). figure 1. schematic presentation of therapeutic and diagnostic potential of micrornas in atherosclerosis, blood pressure regulation and hypertension, endothelial function, inflammation and lipid profile. mir – microrna; bp – blood pressure. seemedj 2022, vol 6, no 1 micrornas and hypertension 55 southeastern european medical journal, 2022; 6(1) these highly stable regulators are primarily found in human serum and saliva (a total of 1881 mirnas identified in human genome) and they can reduce gene expression post transcription by binding to the 3’ untranslated region (utr) of a messenger rna (mrna) leading to repression of translation and/or degradation of target (2,7). lately, efforts are being made regarding investigation of therapeutic use of mirnas in a wide variety of diseases, including cardiovascular disease, immune disorders, rheumatoid arthritis and cancer (8). the fact that mature mirnas are highly conserved and short, makes them perfect for therapeutic manipulations towards modulation of cellular pathways and networks (9). there are two possible proposals for such actions: (1) therapeutic restoration of mirna activity/functionality; and (2) inhibition of mirna function by so-called antimirs – antisense oligonucleotides (4,9,10). in order to draw firm conclusions, it is necessary to take into account that there are great variations in mirna expression levels present, depending on their origin (tissue or cell type) and pathological state, which requires further extensive in vivo experiments in appropriate animal models. schematic representation of mirna breakdown regarding therapeutic/diagnostic potential is presented in figure 1. emerging role of non-coding rna in endothelial function and blood pressure regulation hypertension is, alongside diabetes and cancer, the most common chronic disease and leading cause of death in modern society (11). yet, in a large number of affected individuals, the main cause of elevated blood pressure (bp) cannot be determined with certainty. some of the risk factors associated with hypertension include genetic predisposition, advanced age, low physical activity, obesity and overall poor dietary habits characterized by high sodium chloride (nacl) and low potassium intake (12,13). high-salt (hs) diet normally has a suppressive effect on renin-angiotensin system (ras) – a physiological system that regulates bp – and leads to reduced renin, angiotensin ii (ang ii) and aldosterone plasma levels (14,15), inciting endothelial dysfunction (16–18). however, in hypertensive and diabetic patients, ras activation is not suppressed following salt loading, thus salt loading tends to aggravate the cardiovascular risks in those particular individuals (14,19,20). so far, animal studies have shown that ang ii, hs diet and exercise change mirna levels in hypertension (6). several studies listed mir-22 (21), mir-181a (22) and mir-25 (23) as potential therapeutics in stress response, hypertension and vascular remodelling. furthermore, it is necessary to address environmental factors in future research and find out what influence they could potentially have on mirnas expression, especially in case of hypertension and related complications (6). table 1 represents a crosssection of research studies regarding noncoding rna role in endothelial function and bp regulation. non-coding rnas in hypertension treatment currently, there is a lack of evidence supporting a significant role of non-coding rnas in bp regulation and hypertension but it has been frequently mentioned that mirna activity is deregulated in the state of illnesses. mir-21 is among the first identified non-coding rnas (24) and has been suggested by several studies as a biomarker for early atherosclerosis and hypertension (25–27). in a study by kara et al. (2021), significantly increased levels of mir-21 and aldosterone were found in patients with resistant hypertension compared to newly diagnosed hypertensive patients and healthy controls (28). further, higher levels of mir-21 were reported in hypertensive, stroke and atherosclerotic patients compared with healthy controls, while it also negatively correlated with plasmatic levels of enos in hypertensive patients (29–31). renal mir-214-3p has also been mentioned as a contributor to hypertension by directly targeting enos in rats and potentially humans (32,33). seemedj 2022, vol 6, no 1 micrornas and hypertension 56 southeastern european medical journal, 2022; 6(1) table 1. cross-section of research studies regarding non-coding rnas role in endothelial function and bp regulation mirnas study model targets effects reference mir-21 human model – hypertensive, ischemic stroke and atherosclerotic patients rat model – shr rats mice model c57bl/6j, tac mice mt-cytb enos ↑ mir-21 and aldosterone levels in rht group positive correlation with aldosterone, age, office sbp, 24-h abpm all-day sbp; ↑ crp level, plasma mir-21 expression level and cimt in ht group ↓ nox and enos levels in ht group ↑ mir-21 in stroke and atherosclerotic patients (28) (29) (25) (27) (31) mir-214-3p human model ht and hypertensive nephrosclerosis patients rat model – dahl ss rat, ss.13bn26(l26) rat enos upregulated in kidneys of ht patients and hs–fed ss rats ↓ hypertension and albuminuria in ss model following inhibition of mir-214-2p (33) mir-22 rat model – shr rats mice model – cardiac-specific knockout chga tgfβr i ↓ bp following inhibition ↓ cardiac hypertrophy and ↑ fibrogenesis of cardiac fibroblasts in knockout mice ↑ cardiac contractility and function following inhibition (34) (36) (37) mir-181a mice model – bph/2j mice, mir181a knockout ras ↑ bp and salt-sensitivity in knockout mice ↓ bp and renal renin mrna following treatment with mir181a mimic (40) (41) seemedj 2022, vol 6, no 1 micrornas and hypertension 57 southeastern european medical journal, 2022; 6(1) mir-19a human model – ami and cad patients, rat model – dahl ss rat mice model apoe−/− mice, c57bl/6 mice adrb1 hbp-1 ↑ proliferation and arteriogenesis ↓ apoptosis in endothelial cells ↓ atherosclerotic plaques and lipids load in mice fed with high-fat diet following administration of antagonist (1) (83) (47) (84) mir-192-5p human model hypertensive and hypertensive nephrosclerosis patients rat model – dahl ss rat, ss.13bn26(l26) rat mice model – mir-192 knockout atp1b1 ↓ mir-192-5p in hypertension animal model and following hs diet ↑ map following hs diet in antimir-treated l26 model ↑ map, sbp, dbp following hs diet in knockout mice (32) mir-25 human model – diabetic patients rat model – sd rat mice model – wt mice, c57bl/6 mice, db/db mice cdc42 ↑ ras, hypertension, renal dysfunction following inhibition in normal mice ↓ glomerular fibrosis and bp following inhibition in db/db mice (49) mir-451 human model – pah and hcm patients rat model – wistar rat mice model mir-451 knockout tsc1 negatively correlated with mpap, bnp and adma ↓ development of pah in hypoxia-exposed rats following inhibition ↑ development of hcm following down-regulation (51) (52) mir-29a human model – hypertensive patients rat model – shr rat pten/akt /mtor signalling pathway negatively correlated with the glomerular filtration rate, but positively with crp, tgf-β1, and uacr ↓ hypertrophy and associated indices following inhibition (57) (59) seemedj 2022, vol 6, no 1 micrornas and hypertension 58 southeastern european medical journal, 2022; 6(1) mice model – tac mice (85) shr rats – spontaneously hypertensive rats; tac mice – transverse aortic constriction mice; mt-cytb mitochondrially encoded cytochrome b; enos endothelial nitric oxide synthase; hs – high-salt; rht – resistant hypertension; sbp – systolic blood pressure; abpm ambulatory blood pressure monitoring; crp – c-reactive protein ; cimt carotid intima-media thickness test; ht – hypertension; nox – nitric oxide; ss rat – salt-sensitive rat; ss.13bn26(l26) rat – salt-insensitive rat; bp – blood pressure; chga – chromogranin a; tgfβr i transforming growth factor βr i; bph/2j mice – hypertensive mice; ras renin–angiotensin system; ami acute myocardial infarction; cad coronary artery disease; apoe−/− mice – model of atherosclerosis; adrb1 adrenoceptor beta 1; hbp-1 hmg-box transcription factor 1; atp1b1 atpase na+/k+ transporting subunit beta 1; map – mean arterial pressure; dbp – diastolic blood pressure; sd rat – sprague dawley rat; wt mice – wild type mice; c57bl/6 mice – basic background mouse strain; db/db mice – type ii diabetes model; cdc42 cell division cycle 42; pah – pulmonary arterial hypertension; hcm – hypertrophic cardiomyopathy; tsc1 tsc complex subunit 1; mpap mean pulmonary artery pressure; bnp brain natriuretic peptide; adma asymmetric dimethylarginine. seemedj 2022, vol 6, no 1 micrornas and hypertension 59 southeastern european medical journal, 2022; 6(1) the above mentioned mir-22 targets chromogranin a (chga) mrna – a protein expressed peripherally and in the central nervous system (cns). chga influences bp, vasodilatation, insulin sensitivity, and inflammation (34). animal studies report overexpression of chga and polymorphism of untranslated binding region (utr) for mirnas in spontaneously hypertensive rat (shr) animal model (13), which consequently increases the binding of mir-22 (34,35). inhibition of mir-22 caused a decrease in bp of shrs, which potentially makes it a therapeutic agent in terms of navigating hypertension treatment (7,34). furthermore, results show that mir-22 is an essential regulator of cardiac function and remodelling, since its’ genetic ablation suppresses induced cardiac hypertrophy and enhances fibrogenesis of cardiac fibroblasts in murine model (21,36,37). wahlquist et al. (2014) suggested mir-25 inhibition as a treatment strategy for heart failure, since it improves cardiac contractility and function. it is a repressor of cardiac function and has been upregulated in heart failure events in both murine and human model (7,23). mir-181a is another mirna that has a potential influence on bp. it is the most abundant mirna in lymphoid tissue and it regulates t cell function (38). it showcases somewhat anti-inflammatory effect and contributes to adaptive immunity (39). in knock-out mice, deletion of mir-181a led to salt sensitivity and increased bp while it was downregulated in hypertensive murine and human model (40,41). in mice model of hypertension (schlager bhp/2j mouse), treatment with mir-181a mimics resulted in decreased bp and renal renin mrna (6). recently, we published several papers concerning effects of high-salt intake on inflammation and endothelial function and found altered leukocyte activation status followed by advancement of vascular lowgrade inflammation in both animal and human model (16,42), and also impaired microvascular reactivity in healthy individuals (43). langlo et al. (2021) exposed dahl salt-sensitive (dahl/ss) rats to low-salt (ls) and hs diet, with first resulting in mild to moderate hypertension over time, while the latter resulted in severe hypertension following severely increased systolic blood pressure (sbp) (1). out of 145 studied c-mirnas assessed in that study, 68 of them were associated significantly with hypertensive complications and can potentially serve as biomarkers for diagnostic purposes. among others, mir-19a-5p was suggested as a biomarker in cases of hypertensive encephalopathy and endothelial dysfunction (ed), since it was recognized as the main regulator of platelet activation, coagulation and inflammation (44). enhanced expression of cmir-19a was reported in cases of pulmonary arterial hypertension (45), acute myocardial infarction (46), coronary artery disease and atherosclerotic patients (47). baker et al. (2019) reported antihypertensive effects of renal mir-192-5p in animal hypertension models with an emphasis on the role of atp1b1 target genes (32). they noted decreased levels of mir-192-5p in l26 (ss.13bn26; mild hypertension) and dahl/ss rats following hs loading, with more pronounced effect of hs diet in the latter model. furthermore, when treated with antimir-192-5p, l26 rats had increased mean arterial bp (map) following hs diet. similar effect was detected in mir-192 knockout mice where map, systolic (sbp) and diastolic bp (dbp) increased significantly compared to wild type (wt) mice as a response to a hs diet. these results suggest that deletion or a decrease in mir-192-5p levels leads to exaggerated renal damage and hypertension, suggesting protective role of mir192-5p against hypertension development. in another study, high salt loading caused renal and cardiac dysfunction in uninephrectomized sprague dawley rats (sd) to a larger extent compared to normal rats fed a hs diet. this was accompanied by an increase in levels of mir-25, mir-451, mir-155 and a decrease in levels of mir-99 of the heart, with opposite effect on same circulatory mirnas (48). according to liu et al. (2017), mir-25 levels are lower in blood and tissue samples from diabetic patients/animals and cell cultures exposed to glucose when compared to controls (49). they investigated the effect of knock-down/inhibition of mir-25 on seemedj 2022, vol 6, no 1 micrornas and hypertension 60 southeastern european medical journal, 2022; 6(1) bp, among other parameters, since hypertension is associated closely to diabetic nephropathy. in wt mice, such venture resulted in ras activation and hypertension contributing to renal dysfunction. mirna-25 has also been described as an oncogenic mirna and an important regulator in acute myocardial infarction, left ventricular hypertrophy and heart failure (50). expression levels of mir-451 have been cited in literature as a diagnostic reference in pathogenesis of pulmonary hypertension (51– 53), while circulating mir-155 has been positively correlated with bp (both sbp and dbp) and inflammatory markers, with significantly higher expression levels in hypertensive patients compared to healthy controls (54). as suggested by sun et al. (2012), mir-155 acts as a key regulator of cardiovascular functions, since, when overexpressed, it targets endothelial nitric oxide synthase (enos) expression, decreases it and impairs endothelium-dependent vasorelaxation (55). another potential therapeutic target and diagnosis biomarker (e.g. early stages of hypertensive nephropathy) is mir-29, depicted as necessary for both normal endothelial function and its restoration in animals and humans (56–58). for example, hypertensive patients with left ventricular hypertrophy had significantly higher levels of mir-29a compared to patients with hypertension, while anti-mir inhibition in transverse aortic constriction (tac) mice model resulted in suppressed hypertrophy and associated indices (59). alongside mir-29, mir-30a and mir-133 were also assessed for diagnostic accuracy in case of white-coat hypertension, where their expression levels were associated with bp-related parameters and bp monitoring (60). potential of non-coding rnas in endothelial dysfunction treatment in the last decade, there has been a lot of research dealing with therapeutic inhibition of mir-33 in animal models. there are two isoforms of mir-33 present in humans, mir-33a and mir33b, embedded within srebf1 and srebf2 genes (srebp family of transcription factors), while there is only one isoform present in rodents and non-human primates – mir-33a (9,61,62). these serve a purpose in progression of cardiometabolic diseases such as atherosclerosis and obesity and are crucial factors in lipid metabolism regulation, since they are responsible for maintenance of cholesterol, fatty acid and triglyceride homeostasis (62,63). previous studies reported improved lipid profile (increased circulatory hdl-cholesterol levels), mitigated inflammation and decreased formation of atherosclerotic lesions following inhibition or genetic ablation of mir-33 in mice (63–65). similar effects were also detected in case of inhibition by antimir when administered subcutaneously in ldlr-/mice (deficient for ldl receptor) which resulted in reduced plaque size and atherosclerosis regression as well as improved hdl-cholesterol functionality (66,67). on another note, in non-human primates, both normal and metabolic disease model, treatment with mir-33 targeting antimirs resulted in increased hdl-cholesterol levels, while in normal males’ pharmacological inhibition also resulted in decreased vldl triglycerides (68,69). these results suggest potential therapeutic utility of mir-33s in treatment of atherosclerosis, dyslipidaemia and related metabolic disorders. nuclear factor kappa b (nf-κb), a fairly general transcription factor, regulates a variety of biological processes, particularly in stress response and progression of inflammation, both playing large roles in vascular damage and onset of cardiovascular diseases (70,71). downregulation or decreased expression of mir-150 occurs in acute coronary syndrome and correlates with its onset (72). it targets pentraxin3 (ptx3) and negatively regulates it through inhibition of nf-κb signalling pathway, furthermore attenuating vascular remodelling and restoring endothelial cell function. mir-150 has also been associated with therapeutic potential for thrombosis treatment, since its upregulation has an effect on endothelial progenitor cell differentiation and increases angiogenic potential (73). mir-155, a master seemedj 2022, vol 6, no 1 micrornas and hypertension 61 southeastern european medical journal, 2022; 6(1) regulator of inflammation, plays an important role in regulation of endothelial inflammation through targeting nf-κb pathway and suppression of inflammatory factors, since its inhibition results in significant inflammatory response (74,75). another non-coding rna potentially attenuating endothelial inflammation through effects on nf-κb pathway is mir-99 (76). microrna identification and screening choosing the right mirna for diagnostic/therapeutic purposes and identification of key targets responsible for specific phenotype is somewhat of a challenge of its own (77). understanding of phenotypes evoked by certain mirnas requires usage of computational tools (databases of validated mirnas, prediction algorithms) as well as an experimental approach (gene expression analysis and proteomics), in order to perform functional cell-based screenings in both health and disease conditions (77,78). several reviews and research articles listed functional genomics for appropriate validation of critical proteins in biological networks for the purpose of creating prognostic risk models using mirna data (77– 79). eulalio et al. (2015) (77) provided a review of functional cell-based screening technologies for mirna function, aimed at biological processes and illness-related events, including proliferation, signaling, cell maintenance and differentiation. for characterization of mechanisms of action, it is of crucial importance to identify potential key targets for different mirnas. such information can be detained from computational, prediction algorithms based on complementarity between mirnas and target sequences (80–82). several prediction tools for mirna targets have been developed in the last two decades (miranda, miranda-mirsvr, targescan, dianamicrot-cds, mirtarget2, rna22-gui, targetminer, svmicro, pita, rnahybrid), although it should be clarified that despite the predictive power each approach has, there are also limitations and weaknesses calling for future efforts and research towards the upgrade of available tools (79,81). conclusions circulating mirnas could have a potential for early diagnosis of end-organ injury in hypertension and hypertensive emergency. pathway prediction tools elucidate possible mechanisms in hypertensive emergency that may be the subject for further investigations. further mechanistic studies are needed (e.g. with mirna-214-3p and mirna-29, which have recently been shown to be involved in the development of hypertension). however, currently there are several limitations in using mirnas in clinical diagnosis and therapy, such as large number of mirnas as potential biomarkers that require high-throughput technology (e.g. functional screenings) for investigation, followed by mechanistic studies in animals and humans (the latter much more difficult). acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. langlo kar, silva gjj, overrein ts, adams v, wisløff u, dalen h, rolim n, hallan si. circulating micrornas may serve as biomarkers for hypertensive emergency end-organ injuries and address underlying pathways in an animal model. front cardiovasc med. 2021; 7:626699. doi: 10.3389/fcvm.2020.626699 2. javadifar a, rastgoo s, banach m, jamialahmadi t, johnston tp, sahebkar a. foam cells as therapeutic targets in atherosclerosis seemedj 2022, vol 6, no 1 micrornas and hypertension 62 southeastern european medical journal, 2022; 6(1) with a focus on the regulatory roles of noncoding rnas. int j mol sci. 2021; 22:2529. doi: 10.3390/ijms22052529 3. o’brien j, hayder h, zayed y, peng c. overview of microrna biogenesis, mechanisms of actions, and circulation. front endocrinol. 2018; 9:402. doi: 10.3389/fendo.2018.00402 4. ebert ms, sharp pa. microrna sponges: progress and possibilities. rna. 2010; 16:2043– 2050. doi: 10.1261/rna.2414110 5. gallo a, tandon m, alevizos i, illei gg. the majority of micrornas detectable in serum and saliva is concentrated in exosomes. plos one. 2012; 7:e30679. doi: 10.1371/journal.pone.0030679 6. marques fz, charchar fj. micrornas in essential hypertension and blood pressure regulation. adv exp med biol. 2015; 888:215–235. doi: 10.1007/978-3-319-22671-2_11 7. marques fz, booth sa, charchar fj. the emerging role of non-coding rna in essential hypertension and blood pressure regulation. j hum hypertens. 2015; 29:459–467. doi: 10.1038/jhh.2014.99 8. chakraborty c, sharma ar, sharma g, lee s-s. therapeutic advances of mirnas: a preclinical and clinical update. j adv res. 2021; 28:127–138. doi: 10.1016/j.jare.2020.08.012 9. van rooij e, kauppinen s. development of microrna therapeutics is coming of age. embo mol med. 2014; 6:851–864. doi: 10.15252/emmm.201100899 10. garzon r, marcucci g, croce cm. targeting micrornas in cancer: rationale, strategies and challenges. nat rev drug discov. 2010; 9:775–789. doi: 10.1038/nrd3179 11. heart disease and stroke | cdc. 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131:93–98. doi: 10.1016/j.ejps.2019.02.013 84. gao f, kataoka m, liu n, liang t, huang z-p, gu f, ding j, liu j, zhang f, ma q, wang y, zhang m, hu x, kyselovic j, hu x, pu wt, wang j, chen j, wang dz. therapeutic role of mir19a/19b in cardiac regeneration and protection from myocardial infarction. nat commun. 2019; 10:1802. doi: 10.1038/s41467-019-09530-1 85. shi j-y, chen c, xu x, lu q. mir-29a promotes pathological cardiac hypertrophy by targeting the pten/akt/mtor signalling pathway and suppressing autophagy. acta physiol oxf engl. 2019; 227:e13323. doi: 10.1111/apha.13323 1 author contribution. acquisition of data – n.k., i.d.; administrative, technical, or logistic support – i.d.; analysis and interpretation of the data n.k., i.d.; conception and design n.k., i.d.; critical revision of the article for important intellectual content n.k., i.d.; drafting of the article n.k., i.d.; final approval of the article i.d. seemedj 2021, vol 5, no. 1 imported infections versus herd immunity gaps 1 southeastern european medical journal, 2021; 5(1) original article imported infections versus herd immunity gaps; a didactic demonstration of compartment models through the example of a minor measles outbreak in hungary 1 katalin böröcz *1, ákos markovics 2, zsuzsanna csizmadia 1, joseph najbauer 1, timea berki 1, peter németh 1 1 department of immunology and biotechnology, clinical centre, university of pécs medical school, pécs, hungary 2 department of general and physical chemistry, faculty of natural sciences, university of pécs, pécs, hungary *corresponding author: katalin böröcz, borocz.katalin@pte.hu received: feb 1, 2021; revised version accepted: mar 15 2021; published: apr 28, 2021 keywords: mmr, vaccine, humoral antibody, epidemics, seir model abstract introduction: in hungary, where mmr vaccine coverage is 99%, in 2017, a minor measles epidemic started from imported cases due to two major factors – latent susceptible cohorts among the domestic population and the vicinity of measles-endemic countries. suspended immunization activities due to the covid-19 surge are an ominous precursor to a measles resurgence. this epidemiological demonstration is aimed at promoting a better public understanding of epidemiological data. materials and methods: our previous mmr sero-epidemiological measurements (n of total measles cases = 3919, n of mumps cases = 2132, and n of rubella cases = 2132) were analyzed using opensource epidemiological data (antsz) of a small-scale measles epidemic outbreak (2017, hungary). a simplified seir model was applied in the analysis. results: in case of measles, due to a cluster-specific inadequacy of igg levels, the cumulative seropositivity ratios (measles = 89.97%) failed to reach the herd immunity threshold (hit measles = 92–95%). despite the fact that 90% of overall vaccination coverage is just slightly below the hit, unprotected individuals may pose an elevated epidemiological risk. according to the seir model, ≥74% of susceptible individuals are expected to get infected. estimations based on the input data of a local epidemic may suggest an even lower effective coverage rate (80%) in certain clusters of the population. conclusion: serological survey-based, historical and model-computed results are in agreement. a practical demonstration of epidemiological events of the past and present may promote a higher awareness of infectious diseases. because of the high r0 value of measles, continuous large-scale monitoring of humoral immunity levels is important. (böröcz k, markovics á, csizmadia z, najbauer j, berki t, németh p. imported infections versus herd immunity gaps; a didactic demonstration of compartment models through the example of a minor measles outbreak in hungary. seemedj 2021; 5(1); 1-16) seemedj 2021, vol 5, no. 1 imported infections versus herd immunity gaps 2 southeastern european medical journal, 2021; 5(1) introduction testing of acquired immunity and effectiveness of vaccination against infectious diseases has been increasingly important in the design of preventive public health strategies. resurgence in measles cases in the united states and across europe has occurred, including in individuals vaccinated with two doses of the vaccine (1). according to the centers for disease control and prevention (cdc), world health organization (who) and united nations international children’s emergency fund (unicef), measles has already been a global issue and now it has been aggravated by disrupted immunization protocols due to the covid-19 pandemic (2–5). all six who regions have reported disrupted immunization activities, with major adverse effects on routine immunization and mass vaccination campaigns (4). according to cdc reports, in 2020, more than 117 million children were at the risk of missing out on measles vaccines as a consequence of the covid-19 surge (2). measles immunity gaps resulting from suspended immunization activities are an ominous precursor to a measles resurgence (4). in ukraine, one of hungary’s neighboring countries that was already endemic for measles, vaccination has been interrupted in many regions (3). regarding europe, ecdc surveillance data have indicated an exceptionally high number of measles cases in 2018, 2019 and 2020 in eu/eea countries. vaccination remains one of the safest and most effective interventions available in public health for the primary prevention of infectious diseases, resulting in both direct and indirect immunity in individuals vaccinated (herd immunity) (6–8). even though a safe and effective two-dose measles/mmr vaccination schedule has been available in europe since the 1960s, maintaining high vaccine coverage is still difficult, despite the fact that in hungary, the mmr vaccine is mandatory and consequently the vaccine coverage is estimated to be at 98-99%. according to our previous publications (9,10) and in agreement with the results obtained by our colleagues (11), there are latent immunization gaps in certain age (or immunization) clusters of the hungarian population, with predominance of the ~35-45-year-old adults. these are individuals who form a significant portion of the active labor force of the country, for instance health care workers (hcws). between january 2017 and may 2019, there were 76 reported measles cases in hungary (12), 54 of which were reported between 21 february and 22 march 2017 (13). because of the recent outbreaks worldwide, not only of measles, but also mumps and rubella (mmr) infections, and because of waning of immunity over time after vaccination (14–17), the importance of continuous mmr seroepidemiological screening is evident. suboptimal vaccine effectiveness in certain clusters of the population has a negative impact on overall vaccination coverage. small-scale outbreaks suggest that certain measles vaccines – applied during the early phases of the hungarian vaccination history – failed to elicit the desired immunological response. the resulting immunization gap(s) raise the concern of potential further outbreaks (9,11). the 2020 covid-19 outbreak called attention to the importance of mathematical modelling of epidemics (18). based on a reliable model, the timescale and economic impact of the disease can be predicted and preventive countermeasures can be taken (19). through the example of the measles epidemic in makó (2017, southeast hungary), we demonstrated that, in possession of key epidemiological data (e.g. r0 value, estimated vaccination coverage of a given population, number of infected and recovered individuals and duration of the epidemic), a simple open-source mathematical model can give a good approximation of the course of an infection and may provide better general compliance with protective measures. materials and methods experimental work in this seroepidemiological survey, we combined the data from our previous findings with recent measurements, including antiseemedj 2021, vol 5, no. 1 imported infections versus herd immunity gaps 3 southeastern european medical journal, 2021; 5(1) measles, -mumps and -rubella antibody level (igg) determination. measurements were performed on the automated siemens bep 2000 advance® platform (siemens ag, germany), using our self-developed elisa assays validated by well-established commercial kits, as previously described (9,10). indirect immunofluorescent microscopy was used a reference (euroimmun, germany). in case of large-scale seroepidemiological measurements, a serum bank consisting of anonymous patient sera was used (n of total measles cases = 3919, n of mumps cases = 2132, and n of rubella cases = 2132) (ethical license number 2015/5726). nationally representative samples included randomly selected clinical residual samples, with the exclusion criteria of neonates, children under the vaccination age and severely immunocompromised patients. samples were collected from the department of laboratory medicine (university of pécs, clinical centre). serum samples were from all listed age groups participating in this study and they were categorized based on past changes introduced in measles and mmr immunization schedules. age group determination was based on the landmarks in the history of measles and mmr vaccination schedules in hungary (figure 1). human sera were stored in the accredited laboratory of the department of immunology and biotechnology (university of pécs, medical school, pécs, hungary) according to quality assurance criteria (iso 17025). population-level result evaluation and seropositivity ratio assessment was performed in relation to the concept of herd immunity threshold (hit) values (hit measles = 92–95%, hit mumps = 85–90%, hit rubella = 83–86). the study relies on the full virus antigen repertoirebased indirect elisa method. therefore, it must be considered a good surrogate, rather than an absolute correlate marker for immunity – as far as plotkin’s nomenclature is considered normative (20–22). we examined vaccination group-related infectionand vaccine-induced antibody titres using the following software: spss, origin pro, excel. seir model example and input data a small-scale measles outbreak in hungary in 2017 raised questions about the vaccination coverage rate in the country. experimental results supported the theory of ineffective vaccines, as previously mentioned (9). in spite of its limitations, it seemed reasonable to set up a seir model calculation in order to see whether a few percent decrease in effective vaccination could result in a local epidemic. to demonstrate the disease spread in a well-immunized population where latent immunity gaps may be present, input data were based on the data of the 2017 measles outbreak in makó, southeast hungary. the following parameters were used to perform the calculations: population (n): the epidemic was linked to the small-town hospital. during that year, 65 physicians were responsible for medical attendance of the estimated 30,000 inhabitants of makó and the surrounding villages. in our model, a population of n = 400-1,000 people was assumed, including patients, health-care workers and family members. number of infected individuals (i): a total of 29 cases were reported. incubation time and contagious period: the incubation time for measles ranges from 10 to 12 days on average, an infected person can be contagious even 1-2 days before the first characteristic symptoms are visible, up to 4 days after the rashes appear. in our model, the incubation time (tinc) was assumed to be 10 days, whereas the contagious period (tcont) parameters were determined by equations (5) and (6). reproduction rate ranging from 12 to 18 can be found in the literature and both values were tested. the higher value is applicable to communities where no social distancing is present and the ratio of vaccinated or immunized inhabitants is low. in central europe, the use of the lower value seems more rational, although this specific epidemic was kept mainly in a hospital, where circumstances promote the spread of the infection. in this case, the start of seemedj 2021, vol 5, no. 1 imported infections versus herd immunity gaps 4 southeastern european medical journal, 2021; 5(1) the outbreak was defined as the possible first day of the first patient’s infection, while the model was set to stop after the recovery of the last infected person. when it comes to largescale epidemics, a different approach is used. if no new cases are found after a certain period, the outbreak is over. this time period is usually determined by the incubation time, with a calculation method suggested by the who. based on the elisa antibody measurements, it can be assumed that only ~90% of the hungarian population has effective immunization, which is under the theoretical 92-94% of hit. in the model, 90% of seroprevalence was assumed, but lower values were also tested subsequently. no additional vaccinated (v) compartment was created and immunized individuals were treated as recovered. vital dynamics was disregarded due to the short period of the epidemic. death rate was not taken into consideration either, as no fatalities were observed during the hungarian outbreak. calculations were performed using microsoft excel visual basic application (vba), but the graphs were plotted in origin. vba is a built-in feature of the microsoft office suite with several limitations, but its prevalence and the user-friendly computer language makes it suitable for educational purposes. results changes and historical data regarding epidemics in the hungarian measles/mmr vaccination schedule (23–25) have been plotted on a timeline in order to evaluate seroepidemiological data accordingly. figure 1 shows changes in measles and mmr vaccination schedules in hungary since the introduction of the vaccine (1969). high age-specific attack rates characterizing major epidemics (1980-81 and 1988-89) along with 93%-99% of vaccine coverage evidence insufficiencies of the early vaccination program. figure 1. measles and mmr vaccination schedules in hungary (a) vaccination against measles was introduced in hungary in 1969. (b) from 1969 to 1974, a single dose of measles vaccine was administered in mass campaigns to persons aged 9-27 months. (c) after vaccination was implemented, the incidence rate decreased until 1973-74, when large epidemics occurred primarily in unvaccinated 6-9-year-olds. (d) the recommended age for vaccination was 10 months until 1978, when it was changed to 14 months. (e) after the 1980-81 epidemic, persons born between 1973 and 1977, who received vaccine when the recommended age was 10 months, were revaccinated. (f) the 1988-89 epidemic mainly affected persons aged 17-21, who had been targeted to receive vaccine during mass campaigns in the first years of the vaccination program in hungary. after 1989, children were re-vaccinated at the age of 11 with a monovalent measles vaccine in a scheduled manner. also, in 1989, the rubella vaccine was introduced. (g) in 1990, measles-rubella bivalent vaccines were introduced. (h) the administration of the first vaccine at the age of 14 months lasted from 1978 to 1991. also, in 1991, the measles-mumps-rubella trivalent vaccine was introduced. (i) in 1992, the administration of the first mmr vaccine was shifted to 15 months of age. (j) in 1996, the merck mmr ii vaccine (enders’ edmonston seemedj 2021, vol 5, no. 1 imported infections versus herd immunity gaps 5 southeastern european medical journal, 2021; 5(1) strain, live attenuated) was introduced. (k) in 1999, measles-mumps-rubella revaccination replaced the monovalent measles vaccine. also, in 1999, the gsk pluserix vaccine (measles schwarz strain) was introduced. (l) in 2003, the gsk priorix vaccine was introduced. (m) between 2004 and 2005, the merck mmr ii vaccine was used. (n) between 2006 and 2010, the gsk priorix vaccine was in use. (o) starting from 2011, we have been using a sanofimsd product, mmrvaxpro (measles virus enders’ edmonston strain, live, attenuated), for vaccination and revaccination of children. gsk priorix is still on the market, commonly used for vaccination in adulthood. (p) between january 2017 and december 2019, there were 76 reported measles cases in hungary (according to ecdc surveillance reports). (source of information: mmwr weekly october 06, 1989 / 38(39); 665-668, international notes measles – hungary, http://www.vacsatc.hu, https://www.ecdc.europa.eu) figure 2 shows the age or vaccination groupspecific seropositivity and seronegativity ratios for measles, mumps and rubella. the lowest seropositivity ratios in terms of anti-measles antibody titres (igg) were observed in the groups ‘vaccinated between 1969-1977’ (87.56%) and ‘vaccinated between 1978-1987’ (78.48%). these results are further confirmed by the abovementioned vaccine insufficiencies of the relative periods, described in figure 1. regarding the mumps and rubella seroepidemiological survey, in terms of humoral antibody levels, all vaccination groups satisfied the requirements necessary for the achievement of herd immunity. (a) measles (b) mumps (c) rubella figure 2. measles, mumps and rubella seropositivity ratios according to vaccination groups age / vaccination groups: (i) individuals born before 1969. (ii) individuals vaccinated between 1969 and 1977. (iii) individuals vaccinated between 1978 and 1987. (iv) individuals vaccinated between 1988 and 1990. (v) individuals vaccinated between 1991 and 1995. (vi) individuals vaccinated between 1996 and 1998. (vii) individuals vaccinated between 1999 and 2002. (viii) individuals vaccinated in 2003. (ix) individuals vaccinated between 2004 and 2005. (x) individuals vaccinated between 2006 and 2010 (xi) individuals vaccinated after 2011. the lowest seropositivity ratio (78.48%) was observed in the anti-measles antibody titres (igg) in the group ‘vaccinated between 1978 and 1987’. in case of measles, mumps and rubella cumulative results, the seropositivity ratios were 89.97%, 91.60% and 92.58%, respectively, as shown in figure 3. due to previously detailed seemedj 2021, vol 5, no. 1 imported infections versus herd immunity gaps 6 southeastern european medical journal, 2021; 5(1) cluster-specific inadequacy of humoral antibody levels, the cumulative anti-measles seropositivity ratios also failed to reach the herd immunity threshold (hit measles = 92–95%). measles mumps rubella overall seropositivity ratio overall seronegativity ratio 𝑆𝑒𝑟𝑜𝑝𝑜𝑠𝑖𝑡𝑣𝑖𝑡𝑦 𝑟𝑎𝑡𝑖𝑜 = 𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒𝑠 − 𝛴 (𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 + 𝑒𝑞𝑢𝑖𝑣𝑜𝑐𝑎𝑙 𝑠𝑎𝑚𝑝𝑙𝑒𝑠) 𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒𝑠 ∗ 100 hit measles = 92–95% hit mumps = 85–90% hit rubella = 83–86% figure 3. overall seropositivity and seronegativity ratios n measles = 3,919; n mumps, rubella = 2,132. in case of measles, mumps and rubella cumulative results, the seropositivity ratios were 89.97%, 91.60% and 92.58%, respectively. the overall ratio of seropositive samples was the lowest in the ‘measles’ group, where it remained under the threshold value. seropositivity ratios were calculated as follows: using the seronegativity ratio of 89.97% (≈ 90%) obtained by the cumulative data representation of anti-measles (igg) antibody levels, the model of possible outcomes of a measles outbreak in a hospital as a function of the vaccination coverage rate was investigated. the results of the vba-based seir model of the 2017 epidemic in hungary are summarized in table 1. three parameters – population of the sample, ratio of immunized individuals and reproduction rate of the virus – were set to different values. the effect of these adjustments was investigated and changes in the number of measles cases and timescale of the epidemic were observed. table 1. seir model results for the 2017 measles epidemic in makó, hungary 10% 90% 8% 92% 7% 93% population of the sample (n) ratio of immunised (%) total number of measles cases duration of epidemic 𝑹𝟎 = 𝟏𝟖 1000 90 73 6 months 400 90 29 4 months 400 80 78 3 months 150 80 29 2.5 months 𝑹𝟎 = 𝟏𝟐 1000 90 2 6 days 400 90 2 6 days 400 80 70 4 months 150 80 26 3 months empirical values ? 90 29 2 months seemedj 2021, vol 5, no. 1 imported infections versus herd immunity gaps 7 southeastern european medical journal, 2021; 5(1) at r_0=18 and n = 1000, assuming 90% effective vaccination, 100 susceptible individuals can be found in the population. the model estimates a total number of infected persons at 74 and the duration of the epidemic at half a year, which is more than double of the real values. by setting the population at n = 400, 30 infected individuals and 4 months were given by the model. this way the number of infected persons corresponds to the actual clinical data, but the duration is still longer compared to empirical findings. timescale of the epidemic can be compressed by increasing the proportion of susceptible people. if the vaccination coverage rate is changed from 90% to 80%, the duration of the epidemic is reduced to 3 months, but the total number of infected individuals becomes higher. based on this anomaly, it can be presumed that the total number of involved population might be even lower than 400. unfortunately, the results of the contact tracing procedure were not available for a better approximation. an acceptable correspondence between the model calculations and the clinical data was observed by assuming n = 150 and 80% of vaccination coverage as input parameters. the results – 30 infections in a two-month period – are close to the official values. for a better comparison, modelling with r_0=12 was also performed. the less contagious the virus, the fewer cases are found. using this lower reproduction rate, only isolated cases can occur at 90% of vaccination coverage (which is a value that resembles the hit). by decreasing the vaccination rate, the number of cases increases and the timescale is shortened, similarly to previous test examples. discussion mmr vaccination in hungary in hungary, mmr vaccine is mandatory. a singledose, live-virus combined measles-mumpsrubella (mmr) vaccine is used to vaccinate infants of ≥15 months of age. a reminder vaccine is given to sixth year primary school students (~11 years of age). priorix (gsk), priorix-tetra (gsk), proquad (merck) and the m-mrvaxpro (msd pharma) vaccines are currently used in hungary for vaccination of children (at 15 months and 11 years of age) and for adults (62). the vaccines contain live attenuated viruses (26). regarding insufficient cumulative anti-measles seropositivity levels, we would like to emphasize that potential gaps in the population-level humoral immunity (igg) are attributable to early vaccination periods and are not a general phenomenon relative to the current immunization practices. the susceptibility of certain cohorts is likely attributable to the thermal instability of the historical leningrad-16 vaccine, inefficient seroconversion owing to vaccination at a premature age (e.g. 9 months of age) and the questionable efficiency of the inoculum itself (9, 11, 25, 29, 30, 31). the 2017 measles outbreak in makó was provoked by imported cases. some of our bordering countries are still endemic for measles (27–30). supplementary figure 1 shows the european measles cases in the time period relative to the epidemics in makó and szeged. covid-19 is increasing the risk of measles outbreaks. according to cdc global measles outbreak reports of january 2021, 41 countries may postpone their measles campaigns for 2020 or 2021 due to the covid-19 pandemic. this increases the risk of bigger outbreaks around the world (31). seemedj 2021, vol 5, no. 1 imported infections versus herd immunity gaps 8 southeastern european medical journal, 2021; 5(1) supplementary figure 1. european measles cases in the time period relative to the epidemics in makó and szeged (ecdc.europa.eu) between december 2016 and november 2017, numerous measles cases occurred in europe, most of which were reported by romania, one of hungary’s neighbouring countries. source of data: https://www.ecdc.europa.eu/ 2017 measles epidemic in hungary in 2017, according to the data of the national authorities, a total of 76 persons were infected with measles (corrected to 73 laboratory confirmed cases by ecdc surveillance reports). the outbreak in the hospital of the small town of makó involved 29 individuals and lasted from january 2017 to march 2017 (32,33). in order to demonstrate the spread of virus in a wellimmunized population, where despite good vaccination coverage, latent immunization gaps (unprotected, seronegative cohorts) are present, we used an open-source epidemiological report of the hungarian national public health and medical officer service (antsz) (17 march 2017): ‘at the peak of the hungarian measles epidemics during the spring of 2017, 52 cases with measles-specific symptoms were reported. of these, 15 laboratory confirmed cases (national reference laboratory for measles and rubella, national public health institute, budapest, hungary) were registered by 16 march. of these patients, 12 were health care workers (hcws) and two were hospitalized patients. one of them was a foreigner, while the 86 245 86 7 3 137 5 1 13 296 647 389 36 3 19 3.891 0 2 4 0 18 1 43 29 2.257 1 6 157 30 226 8.638 5755 0 2000 4000 6000 8000 10000 12000 14000 16000 austria belgium bulgaria croatia cyprus czech republic denmark estonia finland france germany greece hungary iceland ireland italy latvia lithuania luxembourg malta netherlands norway poland portugal romania slovakia slovenia spain sweden united kingdom total measles cases by notification rate (ecdc) (december 2016 november 2017; cumulated results) laboratory confirmed case cases seemedj 2021, vol 5, no. 1 imported infections versus herd immunity gaps 9 southeastern european medical journal, 2021; 5(1) other one was a patient living in the vicinity of a hcw. the epidemic affected two health care institutions, the hospital of makó and the clinics of the university of szeged. the first measles case was imported in mid-february 2017 to the hospital of makó. the epidemic affected the hospital staff and their contacts. by 17 march, a measles infection was confirmed in case of a patient who was presumed to be the original importer of the virus, in case of 11 hcws and in case of one of the hcw’s contacts. at the time of this report, additional 11 cases (of which seven hcws and three patients’ contacts) were still under investigation. at the clinics of the university of szeged, two persons – a hcw and a patient – fell ill with measles. another 11 persons (six patients and five hcws) were also suspected at the time of the report. following the appearance of the abovementioned measles cases, in csongrád county, a total of 391 people were vaccinated against measles, mumps and rubella (mmr). as the first cases of this period had been revealed, the national chief physician ordered strict monitoring and reporting of suspected measles virus infections. thus, another 15 suspected cases were registered in several other counties. at the time of the report, laboratory testing was still ongoing (12)’. the second group of imported cases was detected at the end of july 2017 in nyíregyháza, szabolcs-szatmár-bereg county, hungary (11). six unvaccinated romanian children were hospitalised with clinical symptoms of measles. these cases were later laboratory confirmed (national reference laboratory for measles and rubella, national public health institute, budapest, hungary). the subsequent disease spread among two additional hcws (also laboratory confirmed) supports the susceptibility of certain clusters in the hungarian population (11). epidemiological modela didactic representation in this section, we explain the spreading mechanism of infectious diseases for those who are not familiar with the computational background of modelling. to understand the basics of epidemic models, a simplified mathematical interpretation can be used. the spread of a disease can be described by sshaped sigmoid mathematical functions, similar to the well-known ph titration curve, or haemoglobin saturation curves. as infectious diseases spread from human to human, the number of susceptible persons is decreasing over time and it influences the propagation of the pathogenic agent. in the beginning of the outbreak, the damping effect of recovered patients is minimal; the curve is very close to exponential and the number of new cases increases rapidly. at a certain time, a kind of equilibrium follows, daily recoveries can balance new infections and the curve reaches its inflection point. afterwards, in the saturation phase, the epidemic slows down and at the end, no new cases are found and the vast majority of the population has recovered (figure 4). the curves represented in figure 4 are a graphic interpretation of a commonly used method for epidemic modelling – the compartment model. in this model, the population is divided into compartments – well-defined categories based on their epidemiological properties. in a compartment, all individuals behave exactly the same, e.g. they are all infected, all vaccinated, all exposed, etc. the simplest among these compartment models is the sir model, where the letters of the acronym stand for susceptible, infectious and recovered. . seemedj 2021, vol 5, no. 1 imported infections versus herd immunity gaps 10 southeastern european medical journal, 2021; 5(1) 0 30 60 90 120 150 180 0 200 400 600 800 1000 p o p u la ti o n time (days) susceptible recovered infected 94% 6% 8% figure 4. sir model curves of a hypothetical epidemic as the disease spreads, the number of susceptible individuals decreases. first they get infected (i), but later on they will progress to the recovered compartment (r). approximately 6% of the population managed to avoid contact with infected individuals. the peak of infections could be observed almost three months after the first case was recorded, affecting 8% of the population at the same time. the progression of an individual in this model is easy to follow, each member of the population progresses from susceptible to infectious to recovered. 𝑆 𝛽 ⇒ 𝐼 𝛾 ⇒ 𝑅 (1) transition between compartments is described probability of transmitting the disease between a susceptible and an infectious person. in other individuals to whom an infectious person can pass the disease per day (18,39,40) for example, if the infection rate is 0.2, it will take five days on average to infect someone. if we assume that the patient is contagious for 10 days, two new infections are expected in this case. the overall efficacy of the epidemic can be described by the number of these secondary infections originated from the primary infection, our first patient. this important parameter is the basic reproduction number (r0). each virus has its own average r0 value – 12-18 for measles and 3.3-5.7 for covid-19, according to the literature. transition into the recovered compartment. for instance, if this rate is 0.1, the contagious period will last for 10 days. from a mathematical perspective, the transitions can be described by the following differential equations, where s, i and r are the number of individuals in the corresponding compartments, while n is the whole population. ds dt = − βis n (2) di dt = βis n − γi(3) dr dt = γi(4) mathematical methods (such as the rungekutta method) are available for solving similar equations, but there is a simpler option. using the built-in features of microsoft excel (or any equivalent spreadsheet application), it is possible to make calculations using an iterative method. instead of solving the equations, the computer performs calculations that follow the daily changes in different compartments. r0 have to be defined. based on the definition of the transition rates, it can be seen that the recovery rate can be determined by the number of contagious days (t_cont). 𝛾 = 1 𝑇𝑐𝑜𝑛𝑡 (5) the basic reproduction number can be given as follows: 𝑅0 = 𝛽 𝛾 (6) let us assume that in a certain population measles can be transmitted from a single person to 12 others (r_0=12) and they stay contagious for 6 days (t_cont=6). in this case: seemedj 2021, vol 5, no. 1 imported infections versus herd immunity gaps 11 southeastern european medical journal, 2021; 5(1) 𝛾 = 1 𝑇𝑐𝑜𝑛𝑡 = 1 6 (7) 𝛽 = 𝑅0𝛾 = 2 (8) incubation time plays an important role in the spread of a disease. in a more sophisticated model (seir model), this can also be taken into consideration. a new compartment for the exposed part of the population can be generated. the susceptible person first gets exposed and will progress to the infectious state only after a certain time. 𝑆 𝛽 ⇒𝐸 𝛼 ⇒𝐼 𝛾 ⇒ 𝑅 (9) the parameter ‘α’ is a new transition rate, which can be determined by the incubation time (t_inc) , similarly to γ: 𝛼 = 1 𝑇𝑖𝑛𝑐 new compartments can be added to the model anytime, such as the compartment m for individuals with maternal immunity or the compartment e for exposed individuals, who are already infected, but not infectious. based on the characteristics of certain infectious diseases, further models have been developed, such as the sis, msir, seir, seis, mseir and mseirs models. the second ‘s’ in the acronym indicates that after the infection, no permanent immunity can be reached and the individuals step to the s compartment again. in other models, the ratio of hospitalization, the ratio of mild and severe cases and epidemiological interventions can be included, with a more complex mathematical background. in the examples described above some important parameters are simply disregarded, although it is possible to perform a more detailed computation. vital dynamics, the natural dynamics of birth and death, can be included by adding two further parameters. it is necessary to mention that compartment models have their well-known limitations and shortcomings. for instance, all individuals in the population are assumed to have an equal probability of coming in contact with others, although society is inhomogeneous from the perspective of social distancing. another drawback is that the traditional compartment model cannot handle uncertainty in model parameters. working with a smaller set of data increases this uncertainty, making predictions unreliable. to overcome this problem, it is usual to calculate the sir model over a few possible values for each parameter. a more complex solution is to use distribution functions instead of single numbers and if real-time data is available (e.g. we are in the middle of a pandemic), a clinical dataset can be utilized to adjust these parameters (36–38). regarding the seir model resembling the 2017 measles outbreak in makó (figure 4), we would like to note that both the simplified mathematical method and the input data were unreliable. with more sophisticated models, many different parameters can be taken into consideration (37,39). despite that fact, the calculated values correspond in order of magnitude to the available data on the epidemic and support the experimental results describing the vaccination gap. model curves using a lower percentage of the population-level anti-measles protection rate are more fitting. this finding may indicate an even lower percentage of effectively vaccinated population than it was found previously (~90%). it is concluded that the importance of seroepidemiological surveys is confirmed by the recent outbreaks of measles, mumps and rubella infections in several countries (14,16,17,40–45). considering the hit values, suboptimal anti-measles seropositivity ratios were detected in certain clusters of the early vaccination era (78.48% of sufficient antimeasles igg antibody titres among individuals vaccinated between 1978 and 1987). this finding, which is in accordance with a recent study published by our colleagues (11) and historical literature data (46), suggests the existence of age-specific immunization gaps in the hungarian population. for mumps and rubella, our preliminary data shows satisfactory immunity levels. nowadays, in our country, the mmr vaccination coverage is ideal due to the seemedj 2021, vol 5, no. 1 imported infections versus herd immunity gaps 12 southeastern european medical journal, 2021; 5(1) mandatory administration of safe and modern trivalent vaccines. nevertheless, dubious immunization practices in some of our neighboring countries, aggravated by the detrimental effect of the covid-19 pandemic and subsequent suspension of measles vaccination campaigns, may facilitate the occurrence of minor importation-related mev outbreaks in susceptible cohorts. using the example of the 2017 measles outbreak in makó, it has been demonstrated that in possession of key epidemiological parameters (e.g. r0 value, estimated vaccination coverage of a given population, number of infected and recovered individuals, duration, etc.), a simple seir model can give a good approximation regarding the course of an infection. we believe that awareness may significantly reduce the extent of an epidemic (38,47). in the light of current disquieting epidemiological circumstances, we suggest the introduction of open-access mathematical and epidemiological models into modern natural science education of students. today, online epidemic models are easily available for the public (35,36). practical introduction to these plain calculation models could help students understand the rationale behind epidemiological data. we believe that a practical demonstration of epidemiological events can promote a better understanding of countermeasures and also allow for an easier adaptation to the current epidemiological regulations. limitations of experimental work the diagnostic ability of our assay was calculated based on the results obtained by well-established kits capable of humoral antibody detection, rather than on neutralizing antibody titres that could serve as an absolute correlate of protection (48–50). it is important to emphasize that immunity to measles is a complex orchestration between the cellular and humoral immunity. for this reason, only antibody-based definitions of vaccine success and failure may be misleading, or at least simplistic and incomplete (51). limitations of mathematical modelling input data plays a key role in modelling of epidemics. even when the number of cases is high – like in the 2020 covid outbreak – the confidence of fitting is poor at the beginning of new cases vs. time graph. the first cases are usually unexpected, quarantine and social distancing protocols are not applied yet and if the disease has a low prevalence in the population, the accuracy of the diagnosis might be low. besides that, atypical symptoms can be misleading for physicians. furthermore, statistical values, such as basic reproduction number, incubation and recovery time, depend on other factors, such as social distancing and the health care system. acknowledgement. none. disclosure funding. this work was realized with the financial support of the university of pécs, medical school (pécs, hungary) and the following grants: efop-3.6.1-16-2016-00004, 2019 higher education excellence program (fikp ii pop) and huhr/1901/3.1.1/0032 cabcos3. project no. tkp2020-ika-08 has been implemented with the support provided from the national research, development and innovation fund of hungary, financed under the 2020-4.1.1-tkp2020 funding scheme.” transparency declaration: we declare that we have no commercial or potential competing interests or any financial and personal relationships with other people or organizations that could inappropriately influence our network. references 1. haralambieva ih, kennedy rb, ovsyannikova ig, whitaker ja, poland ga. variability in humoral immunity to measles vaccine: new developments. trends mol med 2015;21(12):789-801. doi: 10.1016/j.molmed.2015.10.005. 2. covid-19’s impact on measles vaccination coverage. available from: https://www.cdc.gov/globalhealth/measles/n seemedj 2021, vol 5, no. 1 imported infections versus herd immunity gaps 13 southeastern european medical journal, 2021; 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[cited 2019 aug 22]. available from: https://reliefweb.int/disaster/ep-2019000017-ukr 50. balbi am, van sant aa, bean ew, jacoby jl. mumps: resurgence of a once-dormant disease. j am acad physician assist [internet]. 2018 may 1 [cited 2021 feb 1];31(5):19–22. available from: https://pubmed.ncbi.nlm.nih.gov/29642091/ 51. takahashi s, sato k, kusaka y, hagihara a. public preventive awareness and preventive behaviors during a major influenza epidemic in fukui, japan. j infect public health. 2017 sep 1;10(5):637–43. 52. liu y, liu z, deng x, hu y, wang z, lu p, et al. waning immunity of one-dose measlesseemedj 2021, vol 5, no. 1 imported infections versus herd immunity gaps 16 southeastern european medical journal, 2021; 5(1) mumps-rubella vaccine to mumps in children from kindergarten to early school age: a prospective study. expert rev vaccines [internet]. 2018 may 4 [cited 2019 may 16];17(5):445–52. available from: http://www.ncbi.nlm.nih.gov/pubmed/294783 47 53. bankamp b, hickman c, icenogle jp, rota pa. successes and challenges for preventing measles, mumps and rubella by vaccination. curr opin virol [internet]. 2019 feb [cited 2019 may 15];34:110–6. available from: http://www.ncbi.nlm.nih.gov/pubmed/308524 25 54. kontio m, jokinen s, paunio m, peltola h, davidkin i. waning antibody levels and avidity: implications for mmr vaccine-induced protection. j infect dis. 2012;206(10):1542–8. 55. ovsyannikova ig, dhiman n, jacobson rm, vierkant ra, poland ga. frequency of measles virus-specific cd4+ and cd8+ t cells in subjects seronegative or highly seropositive for measles vaccine. clin diagn lab immunol [internet]. 2003 may [cited 2019 aug 2];10(3):411– 6. available from: http://www.ncbi.nlm.nih.gov/pubmed/127386 40 1 author contribution. acquisition of data: böröcz k, markovics á, csizmadia z administrative, technical or logistic support: böröcz k, csizmadia z analysis and interpretation of data: böröcz k, markovics á, najbauer j, németh p conception and design: böröcz k, markovics á, csizmadia z, berki t, németh p critical revision of the article for important intellectual content: böröcz k, najbauer j, berki t, németh p drafting of the article: böröcz k, markovics á, csizmadia z, najbauer j final approval of the article: najbauer j, németh p. guarantor of the study: berki t, obtaining funding: berki t, németh p provision of study materials or patients: böröcz k, statistical expertise (statistical analysis of data): markovics á, http://www.ncbi.nlm.nih.gov/pubmed/12738640 http://www.ncbi.nlm.nih.gov/pubmed/12738640 seemedj 2021, vol 5, no. 1 regulation of thrombin activity 47 southeastern european medical journal, 2021; 5(1) review article regulation and dysregulation of thrombin activity 1 daria korolova palladin institute of biochemistry, national academy of sciences of ukraine, kyiv corresponding author: daria korolova, d.korolova@gmail.com received: jan 15, 2021; revised version accepted: feb 25 2021; published: apr 28, 2021 keywords: thrombin, protein c, haemostasis, platelets, endothelium, inflammation, covid-19 abstract thrombin is the enzyme of the haemostasis system that stands at the crossroads between the coagulation cascade and coagulation inhibition by protein c, between plasma coagulation factors and cells involved in haemostasis and between haemostasis and the immune system. allosteric regulation of thrombin and its interaction with various partners in blood plasma and on the surface of the endothelium and platelets provides a wide variety of thrombin functions. the thrombin activity regulation is multifactorial, so a failure of any part of this system leads to serious consequences. an example of this are thrombotic/bleeding complications during endothelial dysfunction, infections, inflammation and uncontrolled treatment. this review aims to summarize current knowledge about thrombin structure, functions and regulation. collected data suggest a crucial role of thrombin in different pathologies accompanied by blood coagulation disorders, in particular diseases causing endothelial dysfunction, such as covid-19. (korolova d. regulation and dysregulation of thrombin activity. seemedj 2021; 5(1); 47-64) seemedj 2021, vol 5, no. 1 regulation of thrombin activity 48 southeastern european medical journal, 2021; 5(1) introduction platelet haemostasis and blood coagulation have important and strong interactions, such as positive and negative feedback loops and surface-bound enzyme complexes formation. thrombin is the resulting enzyme produced by the coagulation cascade. it provides not only clot formation, but also a strong enzymatic feedback activity in procoagulant and anticoagulant reactions and platelet reactivity (1). the main thrombin functions are: 1) turning soluble fibrinogen to fibrin, that is forming the protein-polymer core of the thrombus; 2) activating platelets; 3) activating clotting factors v, vii, viii and xi, which enhance thrombin formation; and 4) limiting its own production by binding thrombomodulin and converting protein c (pc) to activated protein c, which subsequently inhibits clotting factors va and viiia. thrombin functions extend beyond blood coagulation and include vascular functioning (2) and in particular, thrombin regulates inflammation and regeneration (3). through par-receptors, it stimulates monocytes, t lymphocytes, leukocytes, endothelial cells, fibroblasts, tissue macrophages, neutrophils and mast cells (4-6). this way, thrombin mediates the crosstalk between the coagulation system and the adaptive immune system at the sites of vascular injury (6). thrombin also enhances proliferation of endothelial cells, epithelium, fibroblasts, smooth muscle and neuromuscular cells and provides an antiviral response (4-6); participates in the development of malignant tumours (stimulates the adhesive and metastatic capacity of tumour cells and activates angiogenesis in the tumour area); regulates the development and apoptosis of nerve cells and neuralgia in the embryonic period and after birth; and controls the survival of the myoblast, the development of the placenta and the embryo as a whole (2-4, 7, 8). thrombin is acknowledged as the central enzyme of haemostasis because of the numerous and multi-directional functions it possesses. when it is present in the bloodstream in pathological conditions, it directly triggers intravascular thrombosis and is a marker thereof at the same time. this is why understanding molecular mechanisms of thrombin regulation is important for both laboratory diagnostics and correction of imbalance in the haemostasis system. thus, the situation with thrombin studies and application is a bit of a paradox. generation of active thrombin is the main factor of intravascular clotting. however, tests indicating thrombin activity or detecting prothrombin activation products are not used as routine laboratory diagnostic tests. also, thrombin is the main target of anticoagulant therapy and at the same time, it can provide both pro-coagulant and anticoagulant action. only a few research groups are working to find ways to switch thrombin activity and direct it to the anticoagulant pathway. this is a promising direction in the search for a fundamentally new treatment of haemostasis disorders. this article is focused on summarizing current knowledge about the diversification of thrombin functions and substrates selection for the purpose of drawing the attention of medical professionals to the importance of using thrombin-specific tests in routine laboratory diagnostics both with regard to haemostasis disorders and infectious diseases. thrombin sites thrombin is a multifunctional serine protease of the haemostasis system. it cleaves more than 10 substrates, but remains highly specific to each one of them. functional plasticity of thrombin is associated with the presence of effector sites (exosites i and ii), which together with the active site are involved in the recognition and binding of the substrate; action directionality modulated by na+ coordination; and significant conformational mobility and plasticity of the molecule (9, 10). in addition, it should be noted that the multi-directionality of thrombin is ensured by its interaction with different partners, so thrombin is characterized by homotropic seemedj 2021, vol 5, no. 1 regulation of thrombin activity 49 southeastern european medical journal, 2021; 5(1) allosteric regulation. in this context, two populations of thrombin can be determined: free and membrane-bound. the thrombin active site is typical for enzymes of the chymotrypsin family of serine proteases. thrombin substrates have a positively charged amino acid (usually arginine) on the n-terminus in the cleavage bond (11) (figure 1). figure 1. thrombin substrate recognition scheme (based on [11, 12]). hph hydrophobic amino acid residue ”– pro or monoamine monocarbon amino acid residue * only for heparin cofactor ii this position is occupied by leu © korolova, 2021 the active site cleft is formed by two loops around the active site, which are longer than such loops in chymotrypsin (12). the 60-loop is hydrophobic and rigid. it forms a cap over an active site and provides interaction with nterminal hydrophobic substrate residues. the γ ‐loop is more hydrophilic and flexible in nature. it is adjacent to the active site cleft of thrombin, it can contact substrate residues c‐terminal to the scissile bond and can make contacts with the body of the substrate protein. conformational changes in the thrombin molecule and changes in its catalytic activity result in the binding of exosites i and ii of thrombin to the ligands (table 1). this is confirmed by a functional analysis that demonstrates the relationship between the exosites and the position of the γ-loop of the catalytic centre. the γ-loop can block access to the “gap" of the active site. active site environments for the individual binary complexes or the ternary thrombin complex are different, such that each binary and ternary thrombin complex could be expected to display unique catalytic properties. at the same time, there is no linkage between exosites i and ii structures. exosite i is formed by lys36, his71, arg73, arg75, tyr76, arg77, lys109 and lys110; exosite ii by arg93, lys236, lys240, arg101 and arg233 (11). exosite i interacts with the cterminus of the substrate, and exosite ii with the n-terminus of the substrate or cofactor it is suggested that the interaction of ligands with the exosites of thrombin results in the displacement of ions from the exosites. in particular, it is known that the efficiency of interaction with exosite ii is strongly dependent on the concentration of nacl, in contrast to the interaction with exosite i (13, 14). both exosites require peptides with a high percentage of acidic residues, 50 % for exosite i and 72 % for exosite ii, but the hydrophobic content is lower for exosite ii. j. huntington (11) analyzed how thrombin chooses to bind ligands to exosite i or ii. the main difference between the exosites is the ratio of negatively charged and hydrophobic amino acids. all exosite i-interacting peptides have a ratio below two and all exosite iiinteracting peptides have a ratio above two. therefore, exosite ii is the only true anionbinding site of thrombin and exosite i is actually the “apolar-binding exosite”. thrombin can interact with ligands by active site only (low-weight substrate), by active site and one of the exosites or by active site and both exosites (thrombomodulin binds simultaneously to thrombin exosite i through its growth factor domains and to exosite ii through a chondroitin sulphate moiety). such interaction variability provides specific recognition of a large range of substrates and their unique rearrangements provide thrombin enzymatic specificity due to seemedj 2021, vol 5, no. 1 regulation of thrombin activity 50 southeastern european medical journal, 2021; 5(1) such recognition. the role of exosites in substrate recognition may not be equal. in particular, fibrinogen recognition is dominated by exosite i binding and exosite ii plays a secondary role in this process. eventually, fibrinogen successfully binds to exosite i of active-site-blocked thrombin (15). table 1. thrombin ligands and characteristics of interaction ligand bound site kinetic parameters without cofactor cofactor kinetic parameters with cofactor fibrin [87] exosite i kd= 0.52 *10-6m (higher affinity site) kd= 180*10 -6m (lower affinity site) fibrinogen [88] exosite i exosite ii active site kd= 7*10-6 m kcat/km = 13.9 *106m-1s-1 (for fpa release) kcat/km = 4.0 *106m-1s-1 (for fpb release) factor v [89] exosite i exosite ii active site kcat/km = 6.3 *106m-1s-1 factor viii [90] exosite i exosite ii active site km = 12.6 *10-9m factor хііі [59] active site kcat/km = 1.4*105m-1s-1 fibrin (in exosite i) kcat/km = 1.2*107m-1s-1 grib [91] exosite ii kd = 5*10-7-10-8 m par-1 [91, 92] exosite i active site kcat/km = 3*106m-1s-1 gрib kcat/km = 1.5*107m-1s-1 kd=10-9-10-10 m protein с [65, 93] active site kcat/km = 5.6*102 m–1s–1 тм (in exosite i, exosite ii) kcat/km = 5.9*106 m–1s–1 tafi [94, 95] active site kcat/km = 0.96*103 m-1s-1 тм (in exosite i, exosite ii) kcat/km = 1.2*106 m-1s-1 kd = 6.6 * 10-9m thrombomodu lin [24,96] exosite i, exosite ii kd = 1 4.9 * 10-9m antithrombin ііі [97] active site kcat/km = 6.8*103 m-1s-1 heparin kcat/km = 1.2*108 m-1s-1 heparin cofactor іі [20] active site heparin, dermatan sulphate kd = 4*10-8m (heparin) kd =1.9 10-6m (dermatan sulphate) heparin [20] exosite ii kd = 1.1*10-7m (low molecular weight) kd =0.9 10-7m (high molecular weight) dermatan sulphate [20] exosite ii kd = 3.6*10-6m seemedj 2021, vol 5, no. 1 regulation of thrombin activity 51 southeastern european medical journal, 2021; 5(1) an interesting example of the regulation significance of thrombin interaction by exosites is fibrinogen γ’ binding. fibrinogen γ’ is a product of alternative splicing. it has an alternative chain with the final four c-terminal residues replaced with 20 different residues, with a high proportion of negatively charged residues. fibrinogen γ’ has an average plasma concentration from 8 % to 15 %. [16]. fibrinogen γ’ chain carboxyl terminus strongly binds to thrombin exosite ii (16). as a result, fibrinogen γ ’ reduces thrombin inhibition by antithrombin iii (at iii) and heparin cofactor іі (hc ii) and competes for binding to thrombin with factor viii. on the other hand, binding to γ’ fibrinogen reduces thrombin blood circulation. in addition, fibrinogen γ’ forms fibrin clots that are resistant to fibrinolysis. so, even a change in the strength of thrombin exosite ii binding to fibrinogen translates into a dramatically different way of clot formation, its content, density and strength of the fibrin network. it should be noted that there is a competition between cofactors for thrombin exosites binding, which has a significant effect on thrombin’s enzymatic orientation. thrombomodulin (tm) and fibrin have been shown to compete for exosite i thrombin binding. however, thrombin has a 1,000 times greater affinity for tm (kd = 1 nm) compared to fibrin (kd = 1 μm), so it always prefers to bind to tm [13]. as the epitopes of binding of tm and fibrinogen, fibrin and par-1 overlap and the binding of thrombin to tm interferes with its procoagulant functions (2). hirudin peptides and fibrinogen bind competitively to exosite i (15). exosite ii binds to the sulphated region (268-282) of glycoprotein ib (gpib) and heparin with an almost equal affinity, but the thrombin prefers to bind to heparin because its concentration in the intact vascular endothelium is much greater than gpib (13). this balance can be changed by vascular damage, endothelial dysfunction, inflammation or platelet activation. after coagulation, full clotting factor viii activation requires cleavage at arg372, a process involving thrombin exosite ii. the sulphated region of gpib binds to thrombin exosite ii and is responsible for the inhibition of the arg372-ser373 bond cleavage and activation of fviii (17). another regulative site of thrombin is the na+binding site. na+ is octahedrally coordinated with the three water molecules and the three oxygen atoms of the carboxyl groups of thrombin [14, 18]. the effect is exclusively allosteric because the na+-binding site is distant from the residues of the catalytic triad (10). the binding of na+ ions dramatically changes the thrombin’s substrate specificity. the “fast” na+bound form of thrombin actively cleaves fibrinogen, fibrin, clotting factors v and viii and par. the “slow” na+-free form has a high affinity for protein c. the transition from “slow” to “fast” thrombin results in the formation of the arg187:asp222 ion pair, the optimal orientation of asp189 and ser195 for substrate binding, and a significant shift of the side chain of glu192 linked to the rearrangement of the network of water molecules that connect the bound na+ to ser195 in the active site. under physiological conditions, the concentration of na+ is 140 mm. the kd for na+ binding to thrombin is 110 mm, which implies that nearly half of the thrombin molecules generated in vivo from prothrombin are in the na+-free, “slow” form (18). it is suggested that the na+-binding thrombin loop is directly involved in protein c interaction (2, 14). the binding of na+ ions plays an important role in fibrinopeptide cleavage by thrombin, since the fibrinogen aα-chain interacts more closely with the thrombin in the transition condition and, by stabilizing the na+-bound form of thrombin, makes catalysis more effective (18). hypernatremia (na+ plasma concentration > 145 mm) or hyponatremia (na+ plasma concentration < 135 mm), which are the most common electrolyte disorders, are often associated with thrombosis or bleeding. even under physiological conditions, the concentration of na+ in the blood drops drastically close to platelet clot in vivo, proving the importance of na+ in controlling the participation of thrombin and other enzymes (clotting factor xa and protein c) in blood coagulation and thrombosis (18). seemedj 2021, vol 5, no. 1 regulation of thrombin activity 52 southeastern european medical journal, 2021; 5(1) thrombin action during coagulation dissociation constants and second-order rate constants of thrombin are quite different for different partners (table 1). the sequence and speed of cleaving of a substrate depend on the dynamic equilibrium between the concentration of free thrombin, thrombin bound to thrombomodulin, thrombin bound to platelets, thrombin in the antithrombin iii-thrombin complex, etc. the thrombin affinity to serpins (serine protease inhibitors) is not very high, unlike the thrombin affinity to glycosaminoglycans (dissociation constants of 110 nm and 90 nm for low and high molecular weight, respectively (19, 20)). this is why the only thrombin inhibitors – antithrombin iii (atiii) and heparin cofactor ii (hc ii) – use heparin and dermatan sulphate to achieve strong interaction with thrombin. glycosaminoglycans accelerate thrombin inhibition by at iii or hc ii 20,000and 70,000-fold, respectively. they decorate proteoglycans in vascular and extravascular spaces and bind to thrombin exosite ii by ionic forces. thrombin inhibition by serpins themselves in the absence of heparin is insignificant. at the same time, heparin presence advances thrombin inhibition and provides strong suppression of coagulation in resting vessels. blood clotting cascade activation in the initial stage of the activation of blood coagulation, subnanomolar amounts of thrombin activate factors v and viii. both exosites are involved in thrombin recognition of these factors. this is how thrombin promotes the formation of the prothrombinase complex and thus enhances the process of its own formation through positive feedback regulation. as a result, a few thrombin molecules quickly raise thrombin production (13, 21). the picomolar concentration of thrombin also activates factor xi (22). the thrombin-gpib complex can activate fxi on the surface of platelets, where thrombin and fxi are colocalized through separate gpib interactions. factor va also was shown as a cofactor in the activation of fxi by thrombin both in a purified system and in blood plasma and requires phospholipid surfaces (23). but it is not clear whether gpib and fva work together or compete. thrombin-cells interaction membrane-bound enzyme complexes are critical for haemostasis (1). complexes bound to the sub-endothelium or activated platelets provide blood coagulation activation, while complexes formed on the surface of the endothelium serve for coagulation inhibition. the density of the binding sites on different surfaces can direct haemostasis and change the status of the coagulation system. for example, about 1 % of fvii circulating in blood is normally activated, but it does not lead to coagulation or exceed the activation threshold because of the absence of a binding surface. while platelet-bound thrombin plays a procoagulant role, thrombin that is bound to endothelial cells provides anticoagulant action (24). endothelium binding thrombin recognizes three partners on the surface of the endothelium. interaction of thrombin with heparan sulphate and thrombomodulin (tm) on the endothelium membrane is rapid, reversible and occurs with a high affinity (table 1), whereas its binding to the membrane protein r-30 is slow, irreversible and occurs with a low affinity (25). this is the way to trigger thrombin activity from coagulant to anticoagulant (figure 2). seemedj 2021, vol 5, no. 1 regulation of thrombin activity 53 southeastern european medical journal, 2021; 5(1) figure 2. enzymatic activity points of free thrombin and membrane-bound enzyme pl – platelet; apl – activated platelet; fg – fibrinogen; fn – fibrin; fdp – fibrin degradation products; tm – thrombomodulin; hep – heparin; thr – thrombin; pc – protein c; apc – activated protein c; epcr – endothelial pc receptor; tafi thrombin-activated fibrinolysis inhibitor.© korolova, 2021 the most striking example of allosteric regulation of thrombin is the change in its enzymatic activity due to its binding to tm. thrombin recognizes tm by the highly charged regions of exosite i and exosite ii. binding of the chondroitin sulphate moiety enhances the affinity of this interaction. the recognition event also involves conformational changes of the thrombin in the “slow” form, mediated by binding of the egf-like domains 5-6 to exosite i (24). protein c interacts weakly with thrombin due to the placement of polar amino acid residues of protein c near the apolar region of the “slit” of the active site of thrombin. tm interacts with thrombin exosite i by the protein part and with thrombin exosite ii by the carbohydrate part. this way, it screens the “unfavourable” amino acid residues of protein c (26). as a result, tm significantly reduces the energy barrier and increases the rate of complex formation [thrombin + protein c] and thrombin activity, in relation to protein c, increases 10,000 times (2, 27). in vivo, the reaction is enhanced approximately tenfold by the endothelial protein c receptor, which binds and localizes pc to the endothelium near tm (figure 2) (28). thrombomodulin is present in an amount of 100,000 copies per endothelial cell. the concentration of free thrombomodulin in large vessels is approximately 0.1-0.2 nm. after interaction with thrombin, its local concentration increases up to 10 nm. it should be noted that in microvessels, the concentration of thrombomodulin reaches 500 nm. this is a prime example of how thrombin-binding sites density can direct haemostasis and provide a significant increase in protein c activity in the microcirculatory system (26). the thrombin-tm complex also activates the carboxypeptidase tafi (thrombin-activated fibrinolysis inhibitor). activated tafi is able to cleave c-terminal lysine residues of fibrin, which typically form fibrinolytic protein binding sites. thus, the activated tafi protects the fibrin clot against lysis [29, 30]. this interaction is mediated by thrombomodulin-exosite i interaction, chondroitin sulphate-exosite ii interaction and tafi-active site interaction. thrombin at high seemedj 2021, vol 5, no. 1 regulation of thrombin activity 54 southeastern european medical journal, 2021; 5(1) concentrations binds to the low-affinity r-30 protein on the endothelium surface (25). thrombin has a low affinity to r-30 and this reaction is slow, but the binding of these proteins is covalent and irreversible. thrombin r-30 complex serves for thrombin uptake, internalization and degradation. platelet binding circulating thrombin levels under 100 pm maintain platelets in an inactivated state. concentration increasing to 1 nm is enough for platelets activation. thrombin partners on the platelet surface are pars (protein activated receptors) and gpib. platelets provide negatively charged surfaces for coagulation factor assembly. prothrombinase complex (factor xa+va assembled on a phospholipid in the presence of ca2+) also works on the platelet surface, so thrombin formation is accelerated by acidic phospholipids and platelet activation is upregulated (figure 2). at low thrombin concentrations, the platelet membrane glycoprotein ib is involved in the interaction between platelets and thrombin. the binding of thrombin to gpib is the responsibility of thrombin exosite ii on one side and the n-terminal domain and negatively charged gpib region on the other. some experimental data (31) suggest that exosite i may also be another site for gpib interaction. the high-affinity thrombin binding site is located in the α-subunit of the gpib (268– 287 amino acid residues), but only a small fraction of the gpib exposed on the platelet surface can bind thrombin and most of the receptors specifically bind only to the von willebrand factor (32, 33). the thrombin-gpib complex can enhance cleavage of gpv, resulting in hyperactivation of platelets. gpib/ix/v signalling pathway mediates pi3k/akt activation and protein phosphorylation (33, 34). it also mediates the increasing intracellular ca2+ in thrombin-activated platelets. the result of thrombin-gpib interaction is the stimulation of energy metabolism (glycolysis and oxidative phosphorylation) (34). gpib can also be a cofactor in case of factor xi (fxi) and par activation by thrombin. thrombin bound to platelet gpib via exosite ii is brought into proximity to par-1 and enhances its activation using exosite i to make contact (11, 13). thrombin signalling in platelets depends principally on pars. thrombin binds par through exosite i and the active site. thrombin cleavage of par is also exosite ii-dependent due to the cofactor effect of gpiba, which accelerates the rate of reaction sixto sevenfold (11, 35, 36). only par-1 and par-4 are exposed on human platelets and endothelium. par-1 is the primary thrombin receptor on platelets, requiring picomolar thrombin concentrations for effective activation, while par-4 cleavage is only relevant at high thrombin concentrations (par-4 contributes to thrombin-induced platelet aggregation at low thrombin concentrations in the range from ~ 0.4 nm to ~ 0.8 nm (37)). par-1 mediates a rapid but transient platelet ca2+ signalling response to thrombin, whereas par-4 mediates a slower, sustained rise, producing the majority of calcium response (38). par-4 also plays a more important role in thrombin generation than par-1 (39). both activation of par-4 and activation of par-1 induce granule release as a feedback mechanism to enhance and stabilize platelet aggregation, with par-1 producing reversible aggregation and par-4 producing irreversible aggregation (37). it is probable that the full response of human platelets requires the formation of a pair of receptors par-1 and par-4 [40]. par-4 does not have a sequence complementary to the thrombin exosites. cleavage of par-4 platelets of humans requires a higher concentration of thrombin (~ 100 times) compared to the splitting of par-1. activation of the par by thrombin begins with binding and cleavage (arg41-ser42) of the n-terminal region of the receptor, which results in the exposure of a new n-terminal region. this amino-terminus serves as a ligand for surfactants, intramolecularly binding to the extracellular portion of the receptor and inducing an intracellular signal (4, 41, 42). thrombin activation is associated with g proteins by par activation. pars signalling seemedj 2021, vol 5, no. 1 regulation of thrombin activity 55 southeastern european medical journal, 2021; 5(1) mediates the activation of phospholipase c isoform β, pi3and rhoa/rho kinases (43-46). pars also mediate increases in intracellular calcium in thrombin-activated platelets. this leads to changes in cytoskeletal actin and platelet surface exposure of fibrinogen-binding integrins iibiiia and activates further signalling and platelet aggregation. the prothrombinase complex converts prothrombin to thrombin by two pathways. initial cleavage at arg320 between the a and b chains generates meizothrombin, which is an active intermediate. the alternative initial cleavage at arg271 cleaves off the gla-domain and the two kringles and generates inactive prethrombin-2. active thrombin is formed as a result of further cleavage of intermediates. both ways take place in vivo (47–50). as previously shown, during the first minute of clotting system activation, the in vitro levels of both thrombin and meizothrombin are equal and can reach 0.8 μm (49). moreover, meizothrombin can bind to the platelet membrane (51), so the local concentration of meizothrombin near the platelet surface may be significantly increased during the initial activation stage. we showed (52) that meizothrombin was able to enhance platelet aggregation induced by adp, collagen or adrenalin. at the same time, being membranebound, meizothrombin is not sensitive to antithrombin iii in contradistinction to thrombin (51). thrombin can also enhance platelet adhesion through cleavage of adamts13, a proteinase responsible for the von willebrand factor processing. since the von willebrand factor is a key platelet adhesion glycoprotein, by inactivating adamts13, thrombin promotes platelet involvement at the site of injury (53). fibrin clot formation the next step of thrombin activity is fibrinogen cleavage. thrombin increasing to µm starts the conversion-of-fibrinogen-to-fibrin thrombin activity (km for fibrinogen cleavage is 7.5 µm). thrombin cleaves four fibrinogen bonds, cleaving two fibrinopeptides: fibrinopeptide a (fpa: 16-amino acid n-terminal peptide from the aα chain) and fibrinopeptide b (fpb: 14-amino acid peptide from the bβ chain) (54). during cleavage of fpa, thrombin binds directly to the aα-chain of fibrinogen via exosite i and the active site. the release of fpa is sufficient for the polymerization of both fibrin and this form of monomeric fibrin desa, which spontaneously polymerizes to form protofibrils. when fpb is cleaved, fibrin desab is formed and lateral association of protofibrils begins. however, the removal of fibrinopeptides b alone does not cause the polymerization of fibrin desb (2, 54, 55). it should be noted that the concentration of thrombin determines the features of the fibrin clot and its resistance to the action of the fibrinolytic system (56). thrombin does not react with fibrin through exosite ii. this allows the release of fibrin from the active site for clot formation and it is the reason why cleavage of fibrinopeptides is not the main thrombin activity. thrombin interacts with the n-terminal e region of fibrin to release fpa and fpb, likely disrupting the interaction of the αc terminus with the e-region and exposing knobs a and b in the e-region, which interact with their specific binding pockets in the dregions of another fibrin molecule, leading to protofibril formation (16). by converting fibrinogen to fibrin, thrombin provides not only clot material, but also its own cofactor. being bound to the fibrin e-region, thrombin uses fibrin as a cofactor for cleavage of factor xiii. factor xiii is bound to the fibrin cterminal d-region. the fibrin monomers spontaneously polymerize and the e-region of one fibrin molecule is located closely to the dregions of two other fibrin molecules, moving factor xiii closer to thrombin. thrombin activates clotting factor xiii by limited proteolysis of its subunit a. factor xiiia cross-links neighbouring fibrin molecules by covalent intermolecular bonds and cross-links inhibitors of fibrinolysis to fibrin (13, 57, 58). the newly generated polymer will provide the cofactor required for 80-fold accelerated factor seemedj 2021, vol 5, no. 1 regulation of thrombin activity 56 southeastern european medical journal, 2021; 5(1) xiii activation (59). this ensures that factor xiiia is generated when it is needed and where it is needed – on the fibrin clot surface. interestingly, purified e-regions of fibrin interact with prothrombin, resulting in thrombin-like active site formation in the prothrombin molecule (60, 61). thus, we can speculate that thrombin-e-region interaction provides not only a thrombin-based approach to factor xiii, but also provides changes in the active site of thrombin and accelerates factor xiii activation. role of thrombin in pathology historically, the procoagulant function of thrombin (the conversion of fibrinogen to fibrin) has been investigated earlier and more extensively. based on the aforementioned facts, we can conclude that thrombin has a much wider range of action. thrombin has a number of functions that can be activated depending on its concentration, localization and accessibility of partner molecules. so, the diversity of thrombin action provides regulation of haemostasis in general and its local concentration influences the thrombus microenvironment and architecture (62). due to a great variety of protein partners, thrombin is a connector between plasma and platelet coagulation, between coagulation and anticoagulation, between haemostasis and the immune system and between endothelium pathology and coagulation disorders. nowadays, thrombin remains a major target of antithrombotic and anticoagulant therapies in cardiovascular medicine. heparins and direct thrombin inhibitors are currently used in the treatment of acute thrombotic complications, but a strategy that inhibits thrombin at the active site reduces not only procoagulant and prothrombotic functions, but also shuts down activity toward the anticoagulant protein c. many researchers have been trying to convert thrombin into a potent and safe anticoagulant for in vivo applications. a new strategy aims at modulating thrombin function, rather than inhibiting it (10, 63). some authors propose that mutant thrombin be used as a safe physiological anticoagulant (64) or apply a fusion protein, where thrombin and the tm domain are connected through a peptide linker (65). the shift of balance in proand anticoagulant action of thrombin becomes crucial for the development of haemostatic pathologies during inflammation and endothelial dysfunction. it should be emphasized that only the native intact endothelium provides the anticoagulant function of thrombin. so, any endothelium dysfunctions lead to triggering thrombin activity toward coagulation, which appears as thrombotic complications during some diseases. high levels of angiotensin ii cause arterial hypertension by a complex vascular inflammatory pathway that requires leukocyte recruitment and reactive oxygen species production and is followed by vascular dysfunction. the resulting vascular inflammation and dysfunction are mediated by the activation of thrombin-driven fxi feedback, independent of factor xii. fxi receptor gpib on platelets is required for this thrombin feedback activation [66]. inhibition of this feedback loop with an antisense molecule against factor xi reduced both vascular pathology and hypertension. at the same time, there are doubts about the importance of feedback activation of factor xi (67). during bacterial and viral infections, there is an interplay between blood coagulation, immune cells and platelets to restrict the dissemination of pathogens within the body. endothelial disturbance switches over coagulation and induces thrombotic complications, excessive inflammation and tissue damage. with regard to potential clinical significance, it is possible that interference with the par1 pathway by direct thrombin inhibitors or par1 inhibitors may increase the risk and severity of viral infection (6). thrombin activity leads to endothelial cell activation in klebsiella infection (bacterial pneumonia). extrinsic pathway generated thrombin mediates fibrin polymerization and platelet‐neutrophil interactions essential for protective immune responses in at least klebsiella pneumonia–derived sepsis [68]. tf is induced in the lung after an h1n1 iav infection in seemedj 2021, vol 5, no. 1 regulation of thrombin activity 57 southeastern european medical journal, 2021; 5(1) mice, which led to thrombin-induced hyperactivation of coagulation (6). at the same time, activated protein c seems to be involved in coagulation regulation during an h1n1 iav infection. hiv infection is associated with increased [thrombin-antithrombin iii]. thrombin mediates the crosstalk between the coagulation system and the adaptive immune system at the sites of vascular injury through increased t-cell motility and production of proinflammatory cytokines during an hiv infection (6, 69). an infection that strongly affects coagulation is the coronavirus disease 2019 (covid-19). it induces an immune response within the endothelium in blood vessels in several organs (70, 71). studies of covid-19 patients demonstrate the presence of fibrin thrombi within distended small vessels and capillaries and extensive extracellular fibrin deposition (72). disseminated intravascular coagulation (dic) has been reported to develop in 70 % of patients who succumb to the infection. d-dimer levels were increased far out of proportion to any abnormalities in the prothrombin time (pt/inr), activated partial thromboplastin time (appt), fibrinogen level, or platelet count; these findings are uncharacteristic of dic as currently understood. hanny al-samkari et al. stated that thrombosis is primarily associated with inflammatory markers, rather than coagulation parameters during a covid-19 infection (73). marco ranucci et al. also demonstrated that coagulation is triggered by the release of il-6 and other cytokines and the consequent release of tissue factor (74). thrombin in laboratory diagnostics in recent times, the most widely used laboratory tests that indicate the imbalance of haemostasis have been the d-dimer test and thromboelastography. d-dimer is the fibrin degradation product appearing in the bloodstream as a result of stabilized fibrin cleavage by fibrinolysis. so, this parameter mainly indicates the existence of stabilized fibrin in the bloodstream and also the balance between coagulation and fibrinolysis (73, 75-77). the thromboelastography characterizes the clotting process in whole blood, providing information on clot formation and lysis overall (78). the thromboelastography testing has expanded to include managing extracorporeal membrane oxygenation therapy, assessing bleeding and assessing hypercoagulable conditions. in addition, thromboelastography platelet mapping has been utilized to monitor antiplatelet therapy (78). in particular, in patients with covid-19, elevated circulating d-dimer levels are associated with mortality (79, 80). but the increasing of d-dimer levels occurs in both thrombosis and bleeding complications, so this parameter cannot be used as a thrombosis marker (81). management of the thrombotic risk associated with covid-19 is complicated by heparin treatment (82) and one of the approaches is modification of the thrombin generation assays conditions by adding heparinase as a heparin neutralizing agent. markers that directly indicate the appearance of active thrombin in the bloodstream are prothrombin fragment 1+2 (f1+2), prethrombin-1 and soluble fibrin. unfortunately, they are not incorporated in the routine laboratory practice. f1+2 is formed during the activation of prothrombin by prothrombinase complex (47), which is why it indicates prothrombinase activity. the appearance of this prothrombin derivative indicates the prothrombin activation to thrombin, so it is a direct method of thrombin detection. the nature of f1+2 makes it a more informative marker of procoagulant changes than clotting tests. marco ranucci et al. were able to show the direct appearance of thrombin by measuring f1+2 in the blood of patients with covid-19 (74). moreover, the f1+2 level was significantly reduced, whereas it increased in non-survivors. another prothrombin derivative is prethrombin1, which appears as a result of prothrombin autolysis by thrombin (9). it is present in the bloodstream in case of intensive production of active non-inhibited thrombin, so it is a result of a strong activation of blood coagulation. a high seemedj 2021, vol 5, no. 1 regulation of thrombin activity 58 southeastern european medical journal, 2021; 5(1) level of prethrombin-1 evidences the danger of intravascular clot formation (73, 83). on the other hand, thrombin activity is a useful predictor of the bleeding risk (73). thrombin acts at the very end of the coagulation cascade and the decrease of its activity is associated with a high risk of bleeding. several studies indicated the correlation between impaired thrombin generation and the severity of the disease (84). the tendency to bleeding was observed when thrombin generation fell below 20 % of the normal range (73). similarly, a correlation between the generation of thrombin and the clinical bleeding phenotype in patients with deficiencies in blood coagulation factors fii, fv, fvii, fx and fix has been observed. in the same way, as prethrombin-1 is a product of thrombin action on prothrombin, the soluble fibrin appears in the bloodstream as a result of the action of small amounts of thrombin on fibrinogen. accumulating in small concentrations that are insufficient for clotting, fibrin desa forms oligomers and macromolecular complexes with fibrinogen, circulating in the bloodstream as non-crosslinked soluble fibrin monomeric complexes. being the direct result of thrombin action, its parameter clearly indicates the activation of blood coagulation even before intravascular clotting (76, 77, 85, 86). thus, the level of thrombin as a diagnostic marker has not been neglected during infectious diseases, including covid-19, and tests that can indicate it directly or indirectly are potentially useful in the prediction of haemostatic abnormalities pertaining to these diseases. conclusion the presence of exosites, active site and na+ site in the thrombin molecule allows thrombin to recognize a large number of substrates specifically. thrombin substrates are important for platelet and fibrin clots formation, for activation and inhibition of the blood clotting cascade, for realization of the endothelial anticoagulant function and for the interaction of haemostasis and the immune system. therefore, thrombin provides wide-scope and multidirectional action in haemostasis. on the other hand, imbalances of various links of haemostasis are reflected in thrombin activity and focus. this is the reason why a significant number of antithrombotic drugs are aimed at inhibiting thrombin activity. at the same time, inhibition of the general and not procoagulant thrombin activity is not the right approach from the point of view of the current understanding of haemostasis. determination of thrombin activity, prothrombin concentration or concentration of prothrombin activation products are not traditional laboratory methods in hemostaseology, despite the fact that thrombin activity is one of the main factors of thrombotic disorders. in our opinion, characterization of the functional state of prothrombin/thrombin should be a required component of everyday laboratory practices. acknowledgement. i am grateful for the help and support of prof. tatyana platonova and dr. volodymyr chernyshenko. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. kuharsky al, fogelson al. surface-mediated control of blood coagulation: the role of binding site densities and platelet deposition. biophysical j. 2001; 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5(2) original article connection between mental disorders and hypertension in patients treated at the psychiatric clinic, clinical hospital centre osijek 1 ivana pavličević tomas 1, dunja degmečić *1 1 psychiatry clinic, clinical hospital center osijek; faculty of medicine, josip juraj strossmayer university of osijek *corresponding author: dunja degmečić, ddegmecic@gmail.com received: sep 29, 2021; revised version accepted: nov 8, 2021; published: nov 26, 2021 keywords: mental disorder, hypertension, depression, comorbidities abstract background: the objective of this study was to assess the type and frequency of psychiatric disorders associated with hypertension and to identify sociodemographic specifics and other comorbid diseases. materials and methods: this was retrospective study to establish if there was a relationship between mental disorders and hypertension in patients hospitalized at the department of psychiatry from january 1, 2020 to august 15, 2021. different mental disorders were correlated with hypertension comorbidity, with the emphasis on disease duration, mental disorder characteristics, diagnostic category such as age and gender, and other comorbidities. results: the study included data from 800 patients hospitalized at the department of acute and biological psychiatry and the department of integrative psychiatry at the psychiatry clinic of the university hospital center osijek. all of them were treated for various mental disorders in the period from january 1, 2020 to august 15, 2021. special emphasis was placed on the patients who had been diagnosed with a mental disorder and hypertension in order to determine how many patients treated for a mental disorder also suffer from hypertension. the research shows that 230 (28.75%) out of 800 patients suffer from both a mental disorder and hypertension. conclusions: the study has shown that almost one third of the respondents treated at the psychiatry clinic for a certain period of time suffer from hypertension. the majority of hypertensive patients were treated for recurrent depressive disorder, alcohol addiction and posttraumatic stress disorder. (pavličević tomas i, degmečić d. connection between mental disorders and hypertension with patients treated at the psychiatric clinic, clinical hospital centre osijek. seemedj 2021; 5(2); 58-67) seemedj 2021, vol 5, no. 2 mental disorders and hypertension in patients treated at the psychiatric clinic 59 southeastern european medical journal, 2021; 5(2) introduction mental disorders, such as anxiety and depression, are risk factors for mortality among cardiac patients, but this issue has received little attention when it comes to hypertensive individuals. research suggests that the correlation between hypertension and total and cardiovascular disease mortality is higher when combined with a common mental disorder (1). other studies have found significant correlation between depression, anxiety, impulsive eating disorders and substance use disorders and the subsequent diagnosis of hypertension. these findings highlight the importance of early detection of mental disorders, as well as of physical health monitoring in patients with those conditions. social phobia and alcohol abuse were more strongly associated with hypertension in males than in females. the correlation between panic disorder and hypertension was particularly apparent in those with earlier-onset hypertension (2). among all mental disorders, depression must be recognized as the leading risk factor for hypertension and cardiovascular incidents. for patients suffering from depression, the risk of developing hypertension depends on: a) biological b) behavioral and c) psychological factors. biological factors certain pathophysiological factors favor faster development of hypertension and other cardiovascular diseases in patients suffering from mental disorders (particularly depression and stress-related disorders): 1. hypothalamic-pituitary-adrenal axis (hpa axis) hyperactivity, which is in the pathogenesis of the mental illness, causes an increase in blood pressure and heart rate, as well as an acceleration of the atherosclerosis process. 2. due to dysfunction of the autonomic nervous system in patients suffering from mental disorders, the activity of their sympathetic nervous system increases, causing catecholamine hypersecretion. this leads to vasoconstriction and enables ventricular irritability. the parasympathetic tone is reduced, which lowers the level of the ventricular ectopy occurrence. in addition to reduced heart rate variability, all of the above significantly increases the risk of sudden death. 3. the state of sub-chronic inflammation caused by hyperactivity of inflammatory cytokinins crp, il 1 and 6 in depressed patients leads to changes in sympathetic tone and influences serotonin and platelet aggregation and fibrinolysis affected by serotonin (3, 4). 4. hypercoagulability, triggered by serotonin and the altered function of platelets affected by serotonin. connecting itself to the platelets via the 5-ht 2a receptors, serotonin participates in the process of platelet aggregation, accelerates vasodilation with the help of preserved endothelium and maintains the flow of blood vessels. in addition to endothelium damaged by atherosclerotic changes, released platelet serotonin causes vasoconstriction and disrupts microvascular circulation. in addition to increased platelet aggregation, a serotoninmediated increased activation of procoagulant factors (fibrinogen, von willebrand factor and factor vii) and the reduction of fibrinolytic activity has been detected in patients suffering from depression (5, 6). behavioral factors people suffering from mental disorders are not physically active enough. they tend to have unhealthy habits: they smoke, drink alcohol, consume food excessively and therefore gain weight, which accelerates hypercholesterolemia and indirectly leads to cardiovascular disease (7). psychological factors persons prone to vascular diseases are known as type a personalities. these are persons who are hyperactive and demanding, unable to relax, and who are aggressive and ambitious. however, from the psychodynamic point of view, they are insecure, with low self-esteem and primarily depressive. if such persons are seemedj 2021, vol 5, no. 2 mental disorders and hypertension in patients treated at the psychiatric clinic 60 southeastern european medical journal, 2021; 5(2) subjected to prolonged stress, it can be manifested both directly (the impact on the cardiovascular system via autonomous nervous system) and indirectly (the impact via the hypothalamic-pituitary-adrenal axis (hpa axis)), which can eventually lead to undesirable outcomes. the symptoms of mental illness may significantly interfere with the course and outcome of a cardiac disease, as well as with the recovery period. the purpose of this study is to analyze the incidence of comorbidity of various mental disorders and hypertension among the patients treated at the department of psychiatry by using the retrospective analysis of medical history. the incidence of comorbidities was compared according to different diagnostic categories of mental disorders, as well as the duration of the disease. incidence of other comorbid somatic diseases has also been studied in those with the aforementioned comorbid mental disorders and hypertension. material/patients and methods the research was conducted at the department of psychiatry at the university hospital center osijek. medical histories and medical records of the hospital information system were used as data sources. respondents were hospitalized at the department of acute and biological psychiatry and the department of integrative psychiatry at the psychiatry clinic. this makes a total of 800 patients with different psychiatric diagnoses, who were hospitalized in the period from january 1, 2020 to august 15, 2021. the following factors were analyzed: the patients’ age and gender, all psychiatric diagnoses for which they were treated, treatment duration and comorbidities. in our study, the emphasis was put on the comorbidity of mental disorders and hypertension, but other somatic disease comorbidities were also evaluated. specific characteristics of the analyzed patient category were observed and recorded using the hospital it system. the criterion for patient selection was the time period in which they were hospitalized at the department of psychiatry (from january 1, 2020 to august 15, 2021). the age and gender of all the patients, as well as their diagnostic categories, disease duration and comorbidities, were all recorded. statistical analysis data collected by reviewing specialist findings were processed using descriptive statistical methods. categorical data are presented in absolute and relative frequencies. numerical data are described by the arithmetic mean and standard deviation in the case of distributions following the normal distribution, and in other cases by the median and limits of the interquartile range. differences or correlations of categorical variables were tested by χ2 test. a specific statistical program spss was used for statistical analysis. significance level was set at p<0.05. results: special emphasis was placed on the patients who had been diagnosed with a mental disorder and hypertension in order to determine how many patients treated for a mental disorder also suffer from hypertension. the results of present study showed that 230 (28.75%) out of 800 patients suffer from both a mental disorder and hypertension. 1.3% of patients who suffered from both mental disorder and hypertension were between 20 and 29 years old; 2.6% ages 30 -39; 10% were 40 49 years old; 35.2% of patients were 50 – 59 years old; 36% were 60 – 69 years old; 12.6% were 70 – 79 years ol, and 2.1% of patients were over 80 years old. other age categories were not recorded. the majority of patients with hypertension comorbidity were between the ages of 60 and 69, whereas the minority were between the ages of 20 and 29. 51.73% of patients with mental disorders and hypertension were women, and 48.26% were men. there is no statistical significance between the sexes. furthermore, the focus of the study was to determine which mental disorders the patients with hypertension comorbidity suffered from (table 1). the highest percentage (30% (n=230) of the patients) suffered from recurrent depressive seemedj 2021, vol 5, no. 2 mental disorders and hypertension in patients treated at the psychiatric clinic 61 southeastern european medical journal, 2021; 5(2) disorder. 17.2% of psychiatric patients with hypertension were treated for alcohol addiction and 16% were treated for posttraumatic stress disorder. there were 14.3% of patients treated for recurrent depressive disorder with psychotic symptoms, and 11.7% of them were treated for schizophrenia. 9.5% of patients with hypertension were treated for psychoorganic symptoms and personality disorders, 7.3% of them were treated for bipolar affective disorder, 6.9% for first depressive episode and 4.7% for schizoaffective disorder. 3.4% of patients with hypertension were treated for mixed anxiety and depressive disorder, 3% for generalized anxiety disorder (gad), 2.6% for inorganic psychotic disorder, 2.2% for adjustment disorder, 1.7% for delusional disorder and dementia. finally, the smallest number of patients suffering from hypertension were treated for first psychotic reaction (1.3%). table 1. all psychiatric diagnoses of the patients with hypertension icd-10 description n % diagnosis f01-f09 mental disorders due to known physiological conditions f03 unspecified dementia 4 1.7 f06 other mental disorders due to known physiological condition 22 9.5 f10-f19 mental and behavioral disorders due to psychoactive substance use f10 alcohol related disorders 41 17.8 f20-f29 schizophrenia, schizotypal, delusional, and other non-mood psychotic disorders f20 schizophrenia 27 11.7 f22 delusional disorders 4 1.7 f23 brief psychotic disorder 3 1.3 f25 schizoaffective disorders 11 4.7 f28, f29 other or unspecified psychotic disorder not due to a substance or known physiological condition 6 2.6 f30-f39 mood [affective] disorders f31 bipolar disorder 17 7.3 f32 depressive episode 16 6.9 f33 major depressive disorder, recurrent 71 30 f33.3 major depressive disorder, recurrent, severe with psychotic symptoms 33 14.3 f40-f48 anxiety, dissociative, stress-related, somatoform and other nonpsychotic mental disorders f41.1 generalized anxiety disorder 7 3 f41.2 mixed anxiety and depressive disorder 8 3.4 f43.1 post-traumatic stress disorder (ptsd) 37 16 f43.2 adjustment disorders 5 2.2 f60-f69 disorders of adult personality and behavior f60 specific personality disorders 22 9.5 https://www.icd10data.com/icd10cm/codes/f01-f99/f01-f09 https://www.icd10data.com/icd10cm/codes/f01-f99/f01-f09/f03https://www.icd10data.com/icd10cm/codes/f01-f99/f10-f19 https://www.icd10data.com/icd10cm/codes/f01-f99/f20-f29 https://www.icd10data.com/icd10cm/codes/f01-f99/f20-f29/f20https://www.icd10data.com/icd10cm/codes/f01-f99/f20-f29/f22https://www.icd10data.com/icd10cm/codes/f01-f99/f20-f29/f23https://www.icd10data.com/icd10cm/codes/f01-f99/f20-f29/f25https://www.icd10data.com/icd10cm/codes/f01-f99/f20-f29/f28https://www.icd10data.com/icd10cm/codes/f01-f99/f30-f39 https://www.icd10data.com/icd10cm/codes/f01-f99/f30-f39/f31https://www.icd10data.com/icd10cm/codes/f01-f99/f30-f39/f32https://www.icd10data.com/icd10cm/codes/f01-f99/f30-f39/f33https://www.icd10data.com/icd10cm/codes/f01-f99/f40-f48 https://www.icd10data.com/icd10cm/codes/f01-f99/f40-f48/f41-/f41.1 https://www.icd10data.com/icd10cm/codes/f01-f99/f40-f48/f43-/f43.1 https://www.icd10data.com/icd10cm/codes/f01-f99/f40-f48/f43-/f43.2 https://www.icd10data.com/icd10cm/codes/f01-f99/f60-f69 https://www.icd10data.com/icd10cm/codes/f01-f99/f60-f69/f60seemedj 2021, vol 5, no. 2 mental disorders and hypertension in patients treated at the psychiatric clinic 62 southeastern european medical journal, 2021; 5(2) depressive disorder and other mental disorders comorbidity were studied in more detail than any other mental disorders monitored and compared to hypertension comorbidity. the study showed that 9.5% of all the respondents had depressive disorder and posttraumatic stress disorder in comorbidity with hypertension, 6% of them suffered from depressive disorder and alcohol addiction, while 1.7% of depressed patients suffered from organic psychosyndrome and hypertension. the study also focused on the treatment period and the criterion included the long-term treatment of the mental disorder. the study showed that 81% of patients suffering from a mental disorder and hypertension had a chronic mental disease. other somatic comorbidities, particularly the most common somatic diseases, were monitored in addition to the mental disorder and hypertension comorbidities. 28.2% of patients suffering from various mental disorders and hypertension also suffered from chronic gastritis, 26.5% of them had diabetes mellitus, 1.8% had hyperlipidemia, and 8.7% had hypothyroidism. of the total number of the respondents with mental disorders and comorbid hypertension, 18.2% suffered only from hypertension and had no other physical disorders, whereas 81.8% also suffered from other physical illnesses (table 2).. table 2. the most common physical comorbidities in patients with hypertension icd-10 description n % diagnosis e11 type 2 diabetes mellitus 61 26.5 e78 disorders of lipoprotein metabolism and other lipidemias 41 17.8 k29 gastritis and duodenitis 65 28.2 e03 other hypothyroidism 20 8.7 discussion the aim of the research was to establish the frequency of hypertension occurring in association with various mental disorders among all patients treated throughout this time period. according to the findings, 28.75% of the hospitalized patients had hypertension, which is consistent with the findings of other international studies. for example, in the study by rantanen et al., the overall prevalence of depressive symptoms among respondents with hypertension was 22.8% (8). furthermore, li et al. found an even higher depression prevalence (29.8%), having conducted a meta-analysis of 10,194 hypertensive respondents in 27 studies using self-assessment scales (9). predominantly conducted were screening studies of patients with hypertension and the level of their association with mood disorders. in this study, the patients’ age and gender were monitored separately among the respondents suffering from a mental disorder and hypertension. the results obtained indicate that the majority of patients (36%) were between the ages of 60 and 69, with the next most prevalent age group being between the ages of 50 and 59 (35.2%). patients aged 20 to 29 had the lowest hypertension suffering ratio. the age groups 70 to 79 and 30 to 39 had a comparable ratio, 12.6% and 10% respectively. the age groups 30 to 39 (2.6%) and above 80 (2.1%) had about the same percentage. patients under the age of 20 were not represented. there was no significant difference in terms of gender distinction: 51.7% were women while 48.3% were men. according to numerous literary sources, there is a difference in the affective disorder occurrence in terms of gender distinction. depression occurs almost twice as often (2:1) in women than in men. the depression https://www.icd10data.com/icd10cm/codes/e00-e89/e08-e13/e11-/e11 https://www.icd10data.com/icd10cm/codes/k00-k95/k20-k31/k29-/k29 https://www.icd10data.com/icd10cm/codes/e00-e89/e00-e07/e03-/e03 seemedj 2021, vol 5, no. 2 mental disorders and hypertension in patients treated at the psychiatric clinic 63 southeastern european medical journal, 2021; 5(2) incidence is estimated to be around 11% in men and 19% in women. the cause for that can be found in hormonal variations, primarily in serotonin, which affect patients’ metabolism. an even greater difference in disorder incidence can be seen in anxiety disorders, with the frequency of anxiety disorders being more frequent in women than in men with a ratio of 3:2, and, according to some studies, of 2:1. as far as bipolar affective disorder and schizophrenia are concerned, it is estimated that the frequency is approximately equal (about 1%) (10). since the predominant diagnostic category of this study was depressive disorder, the obtained results of female predominance were expected. however, in our study a smaller gender difference was observed, which can be attributed to the simultaneous analysis of all the represented diagnostic psychiatric categories. in one study, the most powerful predictor of depression and hypertension comorbidity was the female gender, which was followed by alcohol abuse and obesity. it seems that non-smokers and the moderately physically active people alleviate depression symptoms when compared with the people who are rarely physically active (8). we were particularly interested in diagnostic categories of mental diseases that are in comorbidity with hypertension. of the seventeen monitored diagnostic categories, the highest percentage of psychiatric patients were treated for recurrent depressive disorder (30%), which is also confirmed by other studies. the results of international meta-analysis suggest that when compared with the general population of the same age and gender, depressive patients have a 30% to 50% higher risk of developing cardiovascular disease and hypertension (11, 12). 2 out of 5 patients with coronary artery disease suffer from a clinically significant depressive disorder (12). the mortality rate from cardiovascular diseases is 50% higher among depressive patients when compared to the general population (13). depressive disorder doubles the risk of occurrence of major cardiovascular incidents (such as death caused by coronary heart disease, coronary incidence or myocardial reinfarction, heart failure, myocardial revascularization, fatal and nonfatal stroke) within a two-year-period after acute myocardial infarction (14). the prevalence of depressive disorder is three times higher in patients who have survived acute myocardial infarction in relation to the general population (15). depressed patients have a 35% higher risk of having a stroke than the general population of the same age and gender (16). based on the data from a large population register of sweden, sandström et al. have shown that individuals with hypertension are more likely to suffer from depression than individuals who do not have hypertension (17). according to the us national epidemiological study of monitoring health and nutrition, the comorbidity of hypertension with depression symptoms is associated with a 15% higher relative mortality risk than hypertension without depression symptoms, even after adjusting for lifestyle factors and comorbid diseases (18). higher level of psychological stress in patients treated for hypertension may partly be explained by the hypertension diagnosis (19, 20). the awareness of hypertension may have a labeling effect that causes mental distress, as suggested by hamer et al. (19) on the other hand, michal et al suggested that it could be a consequence of depressed persons’ increased use of health care (20). the second most common diagnostic category in our study was alcohol addiction (17.8%), even though there is a slight variation in terms of the frequency of our study's most common disorder. according to the data obtained in the study, we may conclude that the recurrent depressive disorder is mainly associated with hypertension, while the next most frequent disorder is 1.68 times less frequent. posttraumatic stress disorder was represented in hypertension comorbidity with 16%. the fourth most frequent diagnostic category was recurrent depressive disorder with psychotic symptoms (14.3%). schizophrenia was the fifth most frequent type of mental disorder (11.7%), followed by specific personality disorder (9.5%). the least frequent diagnostic category was acute psychotic disorder, but because it is more seemedj 2021, vol 5, no. 2 mental disorders and hypertension in patients treated at the psychiatric clinic 64 southeastern european medical journal, 2021; 5(2) common in younger patients, this may also explain why it has the lowest prevalence of comorbidity with hypertension. 6.9% of patients suffering from illnesses with hypertension comorbidity were treated for the first depressive episode. we were especially interested in not only the duration of mental disorder and duration of treatment, but also in how the afore-mentioned parameters can affect the occurrence of hypertension and other somatic comorbidities. as regards the duration of the mental disorder in hypertensive patients, 81% of the respondents were receiving longterm psychiatric treatment. their mental illness had lasted for several years, and the majority of them suffered from recurrent depressive disorder. in terms of frequency, alcohol addiction was the second most frequent, followed by post-traumatic stress disorder, both of which were categorized as chronic illnesses. we may conclude that chronic mental illnesses may be a risk factor for the incidence of somatic diseases such as hypertension. because recurrent depressive disorder was identified as the most prevalent chronic illness in patients who also suffered from hypertension, the connections with other mental disorders that occurred in comorbidity with recurrent depressive disorder were additionally analyzed (table 2). the majority of patients (9.5%) suffered from both recurrent depressive disorder and chronic post-traumatic stress disorder. 6% of the patients had a comorbidity of recurrent depressive disorder and alcohol addiction, while 1.7% of patients with recurrent depressive disorder developed psycho-organic symptoms. all of the patients with two chronic mental disorders suffered from hypertension. 81% of patients were in a long-term psychiatric treatment. the data obtained can be linked to the neuroendocrine theories, which are based on the fact that the hypothalamic-pituitary-adrenal axis (hpa axis) is dysregulated in 50% of depressed patients. due to the absence of the negative feedback loop, the hpa axis is hyperactive, resulting in an increase in serum cortisol levels (in the range of 20% to 80%), which, in the biological sense, indicates a continuous state of chronic stress. patients with hpa axis hyperactivity, on the other hand, are more likely to suffer from depression with psychotic symptoms or another mental illness, especially stress-related mental disorders (e.g., posttraumatic stress disorder). thyroid disorders have also been reported in patients with depression (according to some studies, in about 25% of patients) (21, 22). only 18.2% of patients treated at the department of psychiatry had no physical illnesses other than hypertension, which is a modest proportion when compared to 81.8% of those who had multiple concurrent physical diseases. given the large proportion of psychiatric patients with multiple physical comorbidities, the categories of the represented physical diseases were studied separately. the most prevalent diseases were also analyzed separately in terms of their incidence. along with hypertension, chronic gastritis was the most common (28.2%). it can be concluded that almost one third of psychiatric patients suffered from both hypertension and chronic gastritis. the disease with an approximate incidence rate relative to chronic gastritis was diabetes mellitus (26.5%). furthermore, a high percentage of psychiatric patients suffered from metabolic lipid disorders (17.8%). finally, the incidence of hypothyroidism was also studied, and it was found that 8.7% of psychiatric patients with hypertension also suffered from this disease. it is possible to conclude that psychiatric patients belong to a vulnerable group with a high risk of developing chronic physical diseases with a progressive course. according to numerous studies, psychiatric patients are at a higher risk of morbidity and mortality due to physical disorders (23-25). serious and permanent mental disorders can shorten a patient’s life by up to four years when compared to persons who do not have mental disorders. a representative epidemiological study from the 2001-2003 u.s. national comorbidity survey (ncs-r) found that comorbidity between physical and mental diseases was the rule, not seemedj 2021, vol 5, no. 2 mental disorders and hypertension in patients treated at the psychiatric clinic 65 southeastern european medical journal, 2021; 5(2) the exception. (26, 27) more than 68% of adults with a mental disorder reported having at least one physical disorder, and 29% of those with a physical disorder had a comorbid mental disorder. elderly patients and those diagnosed with organic psychosyndromes have been shown to be at the highest risk of comorbid physical illness. (28) in conclusion, a mental disorder is a risk factor for physical disorder and vice versa. in particular, physical illness is one of the strongest risk factors for depression and vice versa (29, 30). an epidemiological study found that the likelihood of being diagnosed with depressive disorder increased with each additional comorbid chronic somatic disease among its respondents (31). according to other studies, depression is comorbid with 26 disease categories and is most prevalent in combination with gastrointestinal disease, stroke, musculoskeletal disease, parkinson's disease, respiratory disease and obesity (32). the study by andres et al. (33) analyzes the association between post-infarction depression in patients with recurrence of acute myocardial infarction, where psychiatric disorders affected the risk of infarction recurrence in the same way that cigarettes, diabetes, and obesity did. several authors reported that specialists could not recognize comorbid somatic diseases in nearly half of all the cases. (34, 35) in some cases, physical diseases lead to psychiatric disorders or they deteriorate the existing symptoms. in addition to mental disorder, the harmful effects of medications or some other treatments can result in serious physical pathology (36). conclusion the study has shown that almost one third (28.75%) of the respondents who were treated at the department of psychiatry for a certain period of time suffered from hypertension. the majority of patients suffering from hypertension were treated for recurrent depressive disorder, alcohol addiction and posttraumatic stress disorder. the least represented diagnostic category was acute psychotic disorder. more than 80% of psychiatric patients with hypertension were in a long-term psychiatric treatment and that fact can be associated with a number of other physical comorbidities they suffered from. acknowledgement. none. disclosure funding. no specific funding was received for this study. competing interests. none to declare references 1. hamer m, batty gd, stamatakis e, kivimaki m. the combined influence of hypertension and common mental disorder on all-cause and cardiovascular disease mortality. j hypertens. 2010; 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44(1):63–75. doi: 10.2190/pm.44.1.e. 34. brugha ts, wing jk, smith bl. physical health of the long-term mentally ill in the community. is there unmet need? br j psychiatry. 1989; 155:777-8. doi: 10.1192/bjp.155.6.777. 35. koran lm, sox hc jr, marton ki, moltzen s, sox ch, kraemer hc, imai k, kelsey tg, rose tg jr, levin lc, et al. medical evaluation of psychiatric patients. i. results in a state mental health system. arch gen psychiatry. 1989; 46(8):733-40. doi: 10.1001/archpsyc.1989.01810080063007. 36. pomeroy c, mitchell je, roerig j, crow s. medical complications of psychiatric illness. washington, dc: american psychiatric publishing, inc; 2002.. 1 author contribution. acquisition of data: pavličević tomas i, degmečić d administrative, technical or logistic support: pavličević tomas i, degmečić d analysis and interpretation of data: pavličević tomas i, degmečić d conception and design: pavličević tomas i, degmečić d critical revision of the article for important intellectual content: pavličević tomas i, degmečić d drafting of the article: pavličević tomas i, degmečić d final approval of the article: pavličević tomas i, degmečić d guarantor of the study: degmečić d provision of study materials or patients: pavličević tomas i, degmečić d statistical expertise (statistical analysis of data): pavličević tomas i, degmečić d seemedj 2022, vol 6, no 1 examination stress, alcohol use and blood pressure 44 southeastern european medical journal, 2022; 6(1) original article investigating the relationship between alcohol use and patterns of blood pressure change due to examination stress among adekunle ajasin university academic staff 1 olasunkanmi rowland adeleke* department of human kinetics and health education, adekunle ajasin university, akungba akoko, ondo state, nigeria. *corresponding author: olasunkanmi rowland adeleke, olasunkanmi.adeleke@aaua.edu.ng received: feb 20, 2022; revised version accepted: mar 27, 2022; published: apr 27, 2022 keywords: blood pressure pattern, alcohol, examination stress, academic staff, stress abstract aim: this study examined the relationship between alcohol use and patterns of blood pressure changes due to examination stress among academic staff at the adekunle ajasin university (aaua). methods: it involved using concurrent mixed methods with quantitative and qualitative approaches. both the questionnaire and the blood pressure and pulse rate reading were used as instruments in data collection. the examined population includes all academic staff of aaua. multistage sampling techniques were used to select participants for the study. in stage one, a simple random sampling technique was used to select five faculties of the university. in stage two, systematic sampling techniques were used to select participants for the study; academic staff in every 5th academic staff office at the selected faculties were selected as a sample frame. two instruments were used in gathering information for this study. the instruments were a self-constructed questionnaire and an electronic sphygmomanometer. data were analyzed using mean and standard deviation at alpha level of 0.05. results: findings revealed that there is a significant difference in the pattern of blood pressure before – f (3, 46) = 4.260, p < 0.05; during – f (3, 46) = 3.570, p < 0.05; and after the examination period – f (3, 46) = 3.131, p < 0.05, based on the respondents’ level of alcohol intake. conclusions: it is recommended that academic staff should be educated on the detrimental health consequences of consuming alcohol to avoid high blood pressure before, during and after the examination period. (adeleke or. investigating the relationship between alcohol use and patterns of blood pressure change due to examination stress among adekunle ajasin university academic staff. seemedj 2022; 6(1); 44-52) seemedj 2022, vol 6, no 1 examination stress, alcohol use and blood pressure 45 southeastern european medical journal, 2022; 6(1) introduction blood pressure can be defined as the physical pressure of blood flowing through the arteries, i.e. the pressure that blood exerts on the inner walls of the blood vessels; it varies in different phases of the cardiac cycle and under different conditions of exertion. high blood pressure accounts for high mortality in the form of coronary heart disease and usually has no warning signs. any form of stress is known to cause definable mental and physiological reactions in the body in the form of alteration of different biological functions, especially the heart rate and blood pressure (1). hypertension is one of the most common health problems with negative consequences, which have devastating effects on human health. in 2016, in the global burden of disease study by risk factors collaborators, high systolic blood pressure was reported as the leading cause of global disease burden in both men and women (2). today, stress has become prevalent in everyday human life, especially among different employees at various job levels. on the one hand, stress is a motivational force and on the other hand, it is a cause of depression. in fact, lack of stress represents the end of life, as there is no enthusiasm for accomplishment of goals. when an employee is at the workplace, there are different stressors present that can have a direct impact on the employee’s performance (3). following this view, work stress emerges when people perceive that they have difficulties in coping with the demands related to work and that their sense of well-being is threatened. many stressors associated with academic staff have been identified. examples include shortage of staff, work overload, too much administrative work, lack of support from superiors and peers, irregular payment of salaries, marking of scripts, lecturing, supervising duty during the examination period, carrying out various research works, etc., as some of the stressors that the academic staff deal with in the university system. stress process is complex and dynamic, fluctuating over the time, places, and persons (4). stress produces a series of distinct biological processes, such as hypothalamic-pituitaryadrenal (hpa) axis activation and associated autonomic nervous system responses (5). many other researchers have found that work stress arises when an individual experiences a demand that exceeds his/her real or perceived abilities to successfully cope with the requirements of the job, resulting in a disorder to his/her emotional and physiological balance (3). examinations are frequently used to evaluate the academic ability of students in a university’s education system. apart from the students, the staff are responsible for smooth running of the examination process, for instance through setting of examination questions, invigilating and marking of scripts to results processing, which represents the academic staff’s multiple roles and responsibilities within the universities. all these responsibilities and many more cause stress, which is a major concern in many, if not all educational institutions around the world. at the university level, administrative duties have increased alongside the escalating demands associated with teaching and research responsibilities. administratively, the lecturers work as counselors, examination officers, staff advisers, department heads, members of various committees at the faculty and department level and they hold many other responsible positions in addition to their teaching duties. thus, the lecturers work under increasing pressure to meet targets set by the university. due to all this workload, some lecturers use alcohol to unwind and also see it as a means of socialization and relaxation. however, this may have a great effect on their blood pressure pattern. for example, it was revealed that examination stress is significantly associated with increased pulse rate, systolic and diastolic arterial pressure (6). this was further affirmed by the finding that high-dose alcohol has a biphasic effect on bp; it decreases bp up to 12 hours after consumption and increases bp > 13 hours after consumption. highdose alcohol increases hr at all times up to 24 hours (7). the rate of alcohol consumption is very high among adults, and two out of every five people are heavy episodic drinkers (8). high blood pressure is particularly prevalent among seemedj 2022, vol 6, no 1 examination stress, alcohol use and blood pressure 46 southeastern european medical journal, 2022; 6(1) blacks and serves as a major risk factor for coronary heart disease, stroke, and heart failure (9). as a result, prevention of high blood pressure early in life is essential for reducing the burden of hypertension for blacks later in life; it likewise serves as a form of primary prevention for heart disease and stroke (8). alcohol consumption is positively associated with bp (10). consequently, alcohol consumption and high blood pressure are among the top five risk factors responsible for the growing global burden of non-communicable diseases (ncd), and are key parts of the world health organization (who) goals to reduce ncd mortality by 25% by 2025. from a public health perspective, both alcohol consumption and high blood pressure are among the most important risk factors for the global burden of ncds (11). a reduction in alcohol consumption will aid a reduction in blood pressure, which has the potential for substantial synergistic health gains in terms of morbidity, mortality, and healthcare costs; yet only about half of the hypertension guidelines worldwide recommend a reduction in alcohol consumption to reduce high blood pressure (12). this would be an important contribution to reaching the goals of the who global action plan for prevention of ncds (13), which stipulates a 10% relative reduction of harmful alcohol use and a 25% reduction in raised blood pressure by 2025 to reduce ncd mortality by 25%. lecturers at the adekunle ajasin university are no exception. due to these reasons, the aim of present study was to establish the relationship between alcohol use and patterns of blood pressure changes due to examination stress among academic staff at the adekunle ajasin university. the main objective of this study is to investigate the relationship between alcohol use and patterns of blood pressure changes due to examination stress among academic staff at the adekunle ajasin university. we hypothesized that alcohol use will have no significant effect on patterns of blood pressure changes due to examination stress among academic staff at the adekunle ajasin university. material and methods descriptive research design of the survey type was used in this study. this method involves observing and describing a subject’s behavior without influencing it in any way. it involves gathering data that describe events and then organizing, tabulating, depicting, and describing the data collection (14). furthermore, it serves to organize findings in order to fit them with explanations, and then to test or validate those explanations (15). the population for this study consisted of all academic staff of the adekunle ajasin university, akungba akoko. multistage sampling techniques were used to select participants for the study. in stage one, a simple random sampling technique was used to select five faculties of the university. in stage two, systematic sampling techniques were used to select participants for the study; academic staff in every 5th academic staff office at the selected faculties were selected as a sample frame. a total of fifty (50) participants were enrolled as the sample size for this study. two instruments were used in gathering information for this study. the instruments are a selfconstructed questionnaire and an electronic sphygmomanometer used to measure the participants’ blood pressure. the questionnaire examined the demographic data of the respondents (age, gender and length of service) and how often the academic staff at the adekunle ajasin university, akungba akoko (aaua), consume alcohol, which is defined in categories as follows – do not consume, occasionally, moderately and a lot. likewise, blood pressure pattern values were measured using a sphygmomanometer and recorded in millimeters of mercury (mmhg) before, during and after the examination period. the pattern of blood pressure distribution is determined in accordance with the categories stated below: normal – below 120 mmhg; elevated – 120– 129/80 mmhg; high blood pressure stage 1 (hbp 1) – 130/80–139/89 mmhg; high blood pressure stage 2 (hbp 2) – 140/90 mmhg and higher. seemedj 2022, vol 6, no 1 examination stress, alcohol use and blood pressure 47 southeastern european medical journal, 2022; 6(1) items in the self-constructed questionnaire were carefully reviewed and submitted to experts in the related field for their review of the instrument; comments and corrections made by experts were carefully incorporated by the researcher. likewise, the sphygmomanometer was calibrated to ensure its adequacy for use. the survey was conducted within a semester that lasted for 4 months. the first application of the instrument occurred one month before the examination period, since not many academic activities were happening at the time. the examination period was a 14-day written exam period, while two weeks after the examination period, another set of data was retrieved and recorded. the survey was also based on continuous alcohol consumption, indicating that before, during or after the examination period, alcohol drinking or lack thereof is a peculiar lifestyle. the instruments were applied by the researcher and two trained research assistants with a 100% guarantee of keeping information confidential. the instrument was applied at each selected faculty at intervals before, during and after the examination period. all questionnaire forms, which also contain information about blood pressure patterns of individual participants, were distributed, retrieved and screened after the last administration. after allowing the respondent to rest for about five minutes, blood pressure was rechecked; the average scores were recorded thereafter. this was repeated when the respondent completed the filling of the questionnaires. ethical consideration informed consent of each subject, ethical and official approval from the local research ethics committee of the department of human kinetics and health education, faculty of education, adekunle ajasin university akungba akoko was obtained for the study and the investigation was performed in accordance with the principles outlined in the study. statistical analysis to determine the reliability of the instrument, a test-retest technique for a two-week interval was employed. this was applied on 5 randomly selected respondents from the target population. these respondents were excluded from the main study. a reliability of 0.89 was obtained using the pearson product-moment correlation coefficient, which was considered adequate for the study. alpha was set at 0.05. the ibm spss version 25 was used for statistical analysis in this study. frequency counts and percentages were used to describe the participants’ information, level of alcohol intake and blood pressure changes due to examination stress. on the other hand, anova was used to test the hypothesis at 0.05 alpha level. systolic blood pressure (mmhg) and diastolic blood pressure (mmhg) records among aaua academic staff based on level of alcohol intake were measured using the t-test. the kolmogorov-smirnov test was used to test for normality of distribution. results table 1 shows that 34 (68%) respondents do not consume alcohol, 12 (24%) consume alcohol at times, 2 (4%) consume alcohol moderately, while 2 (4%) drink a lot. table 1: frequency distribution of respondents by level of alcohol intake frequency percent do not consume 34 68.0 occasionally 12 24.0 moderately 2 4.0 a lot 2 4.0 total 50 100.0 results in table 2 reveal that the blood pressure measurement of 24 (48%) respondents before examination was normal, for 22 (44%) it was elevated, 4 (8%) recorded high blood pressure stage 1, while none was at high blood pressure stage 2. furthermore, the blood pressure measurement of 15 (30%) respondents during examination was normal, for 15 (30%) it was elevated, 17 (34%) recorded high blood pressure stage 1, while none was at high blood pressure seemedj 2022, vol 6, no 1 examination stress, alcohol use and blood pressure 48 southeastern european medical journal, 2022; 6(1) stage 2. likewise, the blood pressure measurement of 22 (44%) respondents after examination was normal, for 22 (44%) it was elevated, 6 (12%) were at high blood pressure stage 1, while none was at high blood pressure stage 2. table 2: frequency distribution of respondents by blood pressure measurement pattern of blood pressure before examination during examination after examination frequency percent frequency percent frequency percent normal (below 120 mmhg) 24 48.0 15 30.0 22 44.0 elevated (120–129/80 mmhg) 22 44.0 15 30.0 22 44.0 hbp 1 (130/80–139/89 mmhg) 4 8.0 17 34.0 6 12.0 hbp 2 (140/90 mmhg and higher) 0 0 0 0 0 0 total 50 100.0 50 100.0 50 100.0 table 3: systolic blood pressure (mmhg) and diastolic blood pressure (mmhg) records of aaua academic staff parameters do not consume alcohol (n = 34) mean ± sd occasionally (n = 12) mean ± sd moderately/a lot (n = 4) mean ± sd total (n = 50) mean ± sd sbp before examinatio n 112.26 ± 21.84 122.75 ± 5.64 125 ± 2.83 115.80 ± 18.88 during examinatio n 119.85 ± 9.76 126.25 ± 8.09 200 ± 4.25 122.40 ± 9.86 after examinatio n 117.18 ± 8.83 122 ± 6.22 129 ± 4.24 119.28 ± 8.67 dbp before examinatio n 70 ± 14.61 72.75 ± 5.91 78 ± 11.31 71.30 ± 12.93 during examinatio n 74.03 ± 8.88 75.42 ± 6.56 115.5 ± 12.34 74.54 ± 8.40 after examinatio n 72.62 ± 8.82 73.50 ± 8.15 77.25 ± 5.31 73.20 ± 8.44 seemedj 2022, vol 6, no 1 examination stress, alcohol use and blood pressure 49 southeastern european medical journal, 2022; 6(1) systolic blood pressure of all individuals when there were no examinations was 115.80 ± 18.884 mmhg; during examinations, it rose to 122.40 ± 9.86 mmhg; while after the examinations, it decreased to 119.28 ± 8.67 mmhg. diastolic blood pressure recorded when there were no examinations was 71.30 ± 12.93 mmhg; during examinations, it was 74.54 ± 8.40 mmhg; while after the examinations, it decreased to 73.20 ± 8.44 mmhg. table 4: anova showing the differences between patterns of blood pressure changes due to examination stress based on alcohol intake alcohol intake n mean std. deviation sum of squares df mean square f sig. average bp measurement before examination do not consume alcohol 34 1.41 .609 between groups 4.348 3 1.449 4.260 .010* occasionally 12 1.92 .515 within groups 15.652 46 .340 moderately 2 2.50 .707 total 20.000 49 a lot 2 2.00 .000 total 50 1.60 .639 average bp measurement during examination do not consume alcohol 34 1.91 .900 between groups 8.068 3 2.689 3.570 .021* occasionally 12 2.58 .793 moderately 2 3.50 .707 within groups 34.652 46 .753 a lot 2 2.50 .707 total 42.720 49 total 50 2.16 .934 average bp measurement after examination do not consume alcohol 34 1.50 .663 3.131 .035* occasionally 12 2.00 .603 moderately 2 2.50 .707 between groups 3.880 3 1.293 a lot 2 2.00 .000 within groups 19.000 46 .413 total 50 1.68 .683 total 22.880 49 p < .05 * – significant, ** – not significant table 4 shows that there is a significant difference in the patterns of blood pressure before (f (3, 46) = 4.260, p < 0.05), during (f (3, 46) = 3.570, p < 0.05) and after the examination period (f (3, 46) = 3.131, p < 0.05), based on the respondents’ level of alcohol intake. discussion at many nigerian universities, administrative duties have increased alongside the escalating demands associated with teaching and research responsibilities. lecturers work as counselors, seemedj 2022, vol 6, no 1 examination stress, alcohol use and blood pressure 50 southeastern european medical journal, 2022; 6(1) examination officers, staff advisers, department heads, members of various committees at the faculty and department level and they hold many other responsible positions in addition to their teaching duties. thus, lecturers work under increasing pressure to meet targets set by the university. due to all this workload, some lecturers use alcohol to unwind and also see it as a means of socialization and relaxation. however, this may have a great effect on their blood pressure pattern. this study revealed that the systolic blood pressure (sbp) of individuals who drink moderately/a lot (125 ± 2.83 mmhg) is higher than of those who drink occasionally (122.75 ± 5.64 mmhg) and those who do not drink alcohol at all (112.26 ± 21.84 mmhg) before examinations commence; sbp of individuals who drink moderately/a lot (200 ± 4.25 mmhg) is higher than of those who drink occasionally (126.25 ± 8.09 mmhg) and those who do not drink alcohol at all (119.85 ± 9.76 mmhg) during examinations; while sbp of individuals who drink moderately/a lot (129 ± 4.24 mmhg) is higher than of those who drink occasionally (122 ± 6.22 mmhg) and those who do not drink alcohol at all (117.18 ± 8.83 mmhg) after the examinations. the findings of this study indicate that systolic blood pressure of all individuals was low when there were no examinations; it increased during examinations and decreased after the examinations. systolic blood pressure was significantly higher in the period during examinations than when there were no examinations (before and after). the study was in line with the findings of (6) that revealed in their study that examination stress is significantly associated with increased pulse rate, systolic and diastolic arterial pressure. diastolic blood pressure (dbp) of individuals who drink moderately/a lot (78 ± 11.31 mmhg) is higher than of those who drink occasionally (72.75 ± 5.91 mmhg) and those who do not drink alcohol at all (70 ± 14.61 mmhg) before examinations commence; dbp of individuals who drink moderately/a lot (115.5 ± 12.34 mmhg) is higher than of those who drink occasionally (75.42 ± 6.56 mmhg) and those who do not drink alcohol at all (74.03 ± 8.88 mmhg) during examinations; dbp of individuals who drink moderately/a lot (77.25 ± 5.31 mmhg) is higher than of those who drink occasionally (73.50 ± 8.15 mmhg) and those who do not drink alcohol at all (72.62 ± 8.82 mmhg) after the examinations. in other words, diastolic blood pressure record when there were no examinations was normal, it increased drastically during examinations and became normal again after the examinations. diastolic blood pressure, which is defined as the minimum pressure during ventricular diastole, with a normal range of 60–90 mmhg and an average of 90 mmhg in adults, was within the normal value during the examination period and the period when there were no examinations (before and after). this could be explained by stimulation of the adrenergic nervous system, which leads to release of catecholamines, in particular noradrenaline at the postsynaptic neuron and adrenaline or epinephrine from the adrenal medulla, which results in activation of α 1, β1 and β2 receptors, consequently elevating systolic blood pressure (16). likewise, the number of hours stayed for examination and invigilation, where many staff members may supervise two to three examinations in a day, long distance covered before reaching the examination center, sitting arrangements of students, counting and marking of exams could be underlying factors as to why systolic blood pressure is elevated. on the other hand, the decrease of systolic blood pressure after examinations have passed can be explained by stating that the decrease results from decrease in peripheral arteriolar resistance and/or cardiac output by a variety of mechanisms at different sites, such as dilatation of resistance vessels, heart pumping against lower resistance, dilatation of capacitance vessels, reduction of venous return to reduce cardiac output, reduction of sympathetic drive to the heart which leads to lower cardiac output, especially in response to examination stress (16). the findings from the analysis of hypothesis one showed that there is a significant relationship between alcohol and patterns of blood pressure changes due to examination stress among academic staff at the adekunle ajasin university. the results also showed that academic staff who consume alcohol moderately had the highest seemedj 2022, vol 6, no 1 examination stress, alcohol use and blood pressure 51 southeastern european medical journal, 2022; 6(1) blood pressure before examinations (mean = 2.50), followed by those who consume it a lot (mean = 2.00) and those who drink alcohol often (mean = 1.92), while people who do not consume alcohol at all (mean = 1.41) had the lowest blood pressure before examinations. during examinations, staff who consume alcohol moderately had the highest blood pressure (mean = 3.50), followed by those who consume it a lot (mean = 2.50) and those who drink alcohol often (mean = 2.58), while people who do not consume alcohol at all (mean = 1.91) had the lowest blood pressure during the examination period. after the examinations, staff who consume alcohol moderately had the highest blood pressure (mean = 2.50), followed by those who consume it a lot (mean = 2.00) and those who drink alcohol often (mean = 2.00), while people who do not consume alcohol at all (mean = 1.50) had the lowest blood pressure after the examinations. thus, academic staff of the adekunle ajasin university, akungba akoko, who consume alcohol a lot and moderately are at greater risk of having high blood pressure than academic staff who consume a little or who do not consume alcohol at all, regardless of whether this occurs before, during or after the examination period, which is in agreement with (7). conclusion there is a significant relationship between alcohol and patterns of blood pressure changes due to examination stress among academic staff at the adekunle ajasin university; those who consume alcohol a lot and moderately are at higher risk of having elevated blood pressure than academic staff who consume a little or do not consume alcohol at all, regardless of whether this occurs before, during or after the period of examination. based on the findings of this study, the following recommendations are made: a) health educators should design effective awareness programs on blood pressure management to educate staff on blood pressure and work stress; b) stress management techniques and a healthy lifestyle through education about health should be made available to the academic staff; and c) academic staff should be educated on detrimental health consequences of consuming alcohol to avoid high blood pressure before, during and after the examination period. acknowledgement. none. disclosure funding. no specific funding was received for this study competing interests. none to declare. references 1 pradhan b, shrestha s, shrestha r, pradhanang s, kayastha b, pradhan p. assessing climate change and heat stress responses in the tarai region of nepal. ind health 2013; 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(1984). print book: english: 2nd ed. englewood cliffs, n. j. prentice –hall, 1984. accessed jan 2022 15. krathwohl dr. methods of educational and social science research: an integrated approach. longman/addison wesley longman, new york, (1993) 789 p. isbn-0-80132029-1. web site: http://longman.awl.com. accessed jan 2022 16. al-sandook ta, alnuaimy karama mt, al saffar mt. effect of stress on arterial blood pressure in dental students. al-rafidain dental journal. 2007; 7(2):118-121 doi: 10.33899/rden.2007.8953 1 author contribution. single author contribution seemedj 2017, vol 1, no. 1 the relationship of saliva microcrystalline characterization… 59 southeastern european medical journal, vol 1, 2017. the relationship of saliva microcrystalline characterization and contractile duration of skeletal muscle in medical students 1 maryana zvir1, andriana beliak1, yaryna pohoretska1, maryana savytska1, oksana zayachkivska1 1 physiology department, danylo halytsky lviv national medical university, lviv, ukraine corresponding author: oksana zayachkivska, md, phd, dsc ozayachkivska@gmail.com introduction the modern lifestyle is characterized by an increasingly sedentary lifestyle, chronic stress overload, and sleep rhythm disorders as a result received: march 14, 2017; revised version accepted: april 23, 2017; published: april 24. 2017 keywords: lifestyle, saliva, microcrystalline, x-ray diffractional powder analysis of intensive usage of various modern gadgets (tablets, smartphones, computer games, etc.), which cause circadian disruption (1,2,3). all these factors lead to additional negative impacts on health, early manifestations of lifestyle diseases, abstract aim: the aim of the study was to analyze the possibility of the diagnostic use of saliva investigation in relation to medical conditions among medical students, and provide a background of non-invasive physiological-based preventive measures for their health outcomes. methods: the study was conducted among 70 students from danylo halytsky lviv national medical university (ukraine), who were asked for general information and interviewed about the subjects of lifestyle risk factors related to physical inactivity, circadian rhythmicity, the use of technical gadgets, and the presence of functional gastrointestinal disorders. their saliva secretion and microcrystallization were evaluated. for detection of the microcrystallines of non-organic origin in saliva, x-ray diffractional powder analysis was used. results: the results of the study indicated that 70% of students have decreased daily skeletal muscle contractile duration, circadian dysfunction and extended time of using gadgets all of which leads to changes in saliva secretion and the microcrystallization of biogeneous substances. about 30% of participants have signs of functional disorders of the upper gastrointestinal tract. conclusion: an integrative view on saliva microcrystalline changes could be a novel diagnostic tool for detection of early health disorders, and maintaining regular skeletal muscle contractile activity and normal circadian rhythmicity is a promising physiological approach to improve health outcomes in young people. (zvir m, beliak a, pohoretska y, savytska m, zayachkivska, o. the relationship of saliva microcrystalline characterization and contractile duration of skeletal muscle in medical students. seemedj 2017;1(1);59-66) seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 60 southeastern european medical journal, vol 1, 2017. osteoporosis, metabolic disorders, obesity, and early aging. prolonged sitting duration and the rapid progress of information technology lead to a sedentary lifestyle of young people, which adversely affects their health and subsequently leads to pathological processes in the body (4,5). the use of modern technological gadgets leads to the desynchronization of circadian rhythms, accompanied by sleep and appetite disturbance, worsening of mood, and initial mental disorders, autonomic nervous system (ans) imbalance, which often stimulates different functional and organic diseases (3). young people are very sensitive to changes of lifestyle and have a tendency to an increased number of mental disorders as cognitive functions decrease, leading to a decline of natural resistance of the organism and initiating a wide range of mental and medical problems, including depression and suicide. all of these lifestyle factors have also been suggested as possible risk factors for upper functional gastrointestinal (gi) disorders (fgid), which are tightly linked to brain-gut interaction (6,7). to prevent health problems in young people and guarantee active aging, the need for the detection of these early risk factors attracted our attention. modern views on saliva show that it can be an important source for the early diagnosis of many diseases and the functional state of our body. moreover, it is known that inorganic and organic components of saliva actively influence the cytoprotection of the esophagogastric region. the effects of normal salivation provide sufficient moisture of the mucous membrane of upper gi part that contains numerous growth factors, like the epidermal growth factor, egf, which provides physiological regeneration, mucin – the formation of mucous-bicarbonate barrier, and specific and non-specific defensive bioregulators, which are crucial for the integrity of the gi epithelial barrier and a general resistance to extreme factors (8). noninvasiveness and simplicity make a salivasampling method attractive for microcrystalline investigations in terms of biogeneous mineral substances. the salivary biogeneous substances were studied using the x-ray diffractional powder analysis (xdpa) technique to determine all mineral phases of biogeneous minerals. recently published data suggests the importance of the inorganic components of saliva, and the quantitative and qualitative state of saliva bioregulators, as determining factors of solid textures that are formed after dehydration (9,10,11). thus, we formed a hypothesis that the investigation of saliva as a non-invasive simple diagnostic tool could be used for detection of the early changes, associated with ans imbalance, caused by physical inactivity (12,13). in the present study, we aimed to provide an integrative view of saliva investigation, the screening of lifestyle risk factors, and the skeletal muscle contractility duration that occurs in healthy medical students. material and methods the study was conducted on a group of 70 participants who were second-year students of the medical faculty from danylo halytsky lviv national medical university (mean age 18.5 years, 38 women and 32 men) with 16 months of an academic lifestyle (during studying). selected students were healthy—without any previous chronic diseases and addictions, including caffeine abuse or family-ancestral diseases— and also possessed good oral health—without dentures or dental restorations—according to the reports of the student’s dental and orofacial examinations by dentists. their daily fluid intake did not exceed 2.5 l. they were eligible to participate in this study. the exclusion criteria were medical students who refused to participate. the students who agreed to participate signed a consent form. the design of the study and the test procedures were approved by the ethics committee of lviv national medical university (15.02.2016; n2). the subjective and objective evaluation of the students was divided into 3 parts. the first part of the study concerned general information: gender, previous general illness, administered medication, addictions, and an anthropometric seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 61 southeastern european medical journal, vol 1, 2017. examination. anthropometric data included height, weight, body mass index (bmi), and age. height was measured with a regular stadiometer, while weight and body composition, using the bioelectric impedance method, were evaluated with electronic scales (omron corporation, kyoto, japan). bmi was calculated using the following parameters: fat content (% of body weight), visceral fat (%), and muscle mass (%). the second part of the study was based on questionnaires: the international physical activity questionnaire (ipaq) (14), the pittsburgh sleep quality index (psqi) (15), 10 closed questions on the usage of technological gadgets, and a determination of the daily time of physical inactivity (sitting time more than 60 minutes without a break), estimated as a mean value after evaluation every 5 days, and finally the assessment of the symptom manifestations of upper fgids through a standard self-report questionnaire, included in the rome iii consensus (7). the third part of study concerned the saliva investigation by the examination of samples of saliva that were collected at the same time, not less than 2-3 hours after eating in the morning (between 9 and 11 a.m.), with a sterile pipette from the bottom of the oral cavity, in the volume of 2 ml into dry sterile test tubes. all women were examined in the estrogen phase of their menstrual cycle. the level of stimulated and non-stimulated saliva secretion was measured. non-stimulated saliva was collected from students in the beginning. later, students were asked to chew gum without sugar for 5 minutes and stimulated saliva was carried out in a similar manner into test tubes. both deposition time and volume of deposited saliva were measured. the collected data made it possible to determine the rate of saliva secretion (ml/min). the morphological type of saliva crystallization was estimated by the dehydration of drops of mixed saliva in the air at room temperature for 24 hours on a sterile glass slide using light microscopic investigation with a leica dm 750/4 microscope and leica dfc 420 digital photo camera, both manufactured in germany (figure 1), and by phase contrast imaging for the evaluation of saliva samples without fixing and staining with leica dm-2500 (switzerland) microscopy and camera leica dft 450 with software application suit version 4.4 microscope leica dm-2500 (switzerland) (figure 2). the evaluation of saliva crystallization was performed blind by two independent investigators and the estimation of the type was based on grading four main types according to shatohina s.n., 2004 (16). the first (1st) type is characterized by clear crystal treadprismatic structures connected between themselves in a fern leaf and evenly placed; the second (2nd) type is characterized by certain tree-crystal or small single crystals of various shapes placed evenly on the field of view in a grid; the third (3rd) type is characterized by isometric-placed irregularly shaped structures; and the fourth (4th) type is characterized by the absence of crystals. the rest of saliva was taken for further tests. for detection of the microcrystallines of nonorganic origin in saliva, x-ray diffractional powder analysis was used. for this samples of saliva were selected with the 4th type of crystallization. the samples were applied on the polymer coat and remained there until drying. the coat with dry substance was fixed and covered with the second coat. experimental intensities and angles reflected from samples were obtained by an automatic diffractometer stoe stadi p (“stoe & cie gmbh”, germany) with a linear position-precision detector. primary processing of the experimental diffraction, the calculation of the theoretical diffraction, and the indexing parameters of unit cells were performed using the software package stoe winxpow and powdercell with the method of comparing x-ray diffraction profiles (18,19). statistical data analysis was performed by calculating means with their standard errors and proportions. differences in means were estimated by the mann-whitney u-test. all data was processed using the statistical package statistica 10.0 (statsoft, tulsa, oklahoma, usa). results among the 70 medical students of lviv national medical university who participated in the study seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 62 southeastern european medical journal, vol 1, 2017. 32 (46%) were males and 38 (54%) females, with table 1. study group’s general information characteristics (n=70). variable results gender male: 32 (45.7%) female: 38 (54.3%) age mean: 18.5 years, range 17-19 years height mean: 171±9 cm, range 156-190 cm weight mean: 63±9 kg, range 47-82 kg bmi mean: 21.5±1.4 kg/m2, range 18-24.5 kg/m2 body composition: fat content visceral fat muscle mass mean: 25.1±6.8%, range 14.2–36.6% mean: 6.14±1.2%, range 3.94–8.73% mean: 29.7±6.8%, range 24.2–44.2% current smoker total: 13 (18.6%) table 2. types of physical activity among medical students (mean values in met min/week). type of activity gender total male female moderate 748.1 545.6 632.4 intensive 938.7 456.5 618.2 walking 984.2 1024.1 992.2 total 2,671.0 2,026.2 2,242.8 figure 1. light microscopy of samples of saliva by the type of microcrystallines in medical students (data from study group); x 120: a – 1st type; b – 2nd type; c – 3rd time; d – 4th type. table 3. characterization and duration of daily physical inactivity among medical students. daily sitting time 6-8 hours/day >8 hours/day 48 (68.6%) 22 (31.4%) total usage of gadgets 2-3 hours per day 4-5 hours per day >5 hours per day 24 (34.2%) 31 (44.3%) 15 (21.5%) usage of gadgets for more than 2 hours without a break never only occasionally a few times per month a few times per week almost every day 8 (11.6%) 12 (17.1%) 18 (25.6%) 21 (30.0%) 11 (15.7%) table 4. differences between distributions of types of saliva between groups of students with and without functional gastrointestinal disorders is statistically significant (p<0.001). type of saliva microcrystalli -nes symptoms of upper functional gastrointestinal disorders no yes n % n % 1 0 0 12 24.5 2 1 4.8 24 49.0 3 8 38.1 11 22.4 4 12 57.1 2 4.1 all 21 100.0 49 100.0 figure 2. micro photos of saliva samples from medical students by phase contrast imaging, ranging to four microcrystallines types: a – 1st type; x200; b – 2nd x200; c – 2nd x400; d – 3rd x200; e – 4th x200; f – 4th x400 (data from study group). seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 63 southeastern european medical journal, vol 1, 2017. a mean age of 18.5. table 1 presents the data for all participants, including age, gender, administered addictions, and anthropometric examination. anthropometric data involved height, weight, bmi, body composition and general characteristics of study group represented in table 1. the physical activity of medical students involved in the study was estimated by ipaq, expressed by arithmetic mean (met min/week), to have reached the value of 2,242.8, with higher values observed in males (2,671.0) than in females (2,026.2). we also estimated the type of activity: moderate, intense, and walking. a larger number of participants confirmed walking as the dominant type of physical activity. walking was the dominant type of physical activity in females (1024.1 vs. 984.2 in males) (table 2). the study assessed the level of physical activity that was calculated from the total physical activity and categorized it into three ranks: low, moderate, and high. a significant variation was observed at each level according to the gender, and the larger differences were in high level of physical activity in males (43.8%) vs. females (23.7%). moderate physical activity was better obtained in females (50.0%), compared to males (34.4%). it was observed that 78.5% of medical students use gadgets 3.6±0.5 hours/day and 21.5% of them spend more than 5 hours/day with technical gadgets. there were no significant differences in gender distribution in the collected data. more than half of participants do not take any breaks when they use computers, leading to an increased daily sitting time figure 3. results of investigation of saliva bio compounds by x-ray powder diffraction analysis: a – tested comparison of packet film and investigated dry precipitate of saliva samples; b – detection of two-phase kcl microcrystallines and undetected compound; c – standardization with theoretical diffractogram kcl; d – kcl detection with small traces of sodium. seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 64 southeastern european medical journal, vol 1, 2017. characterized as a physically inactive lifestyle (table 3). we also screened sleeping patterns by psqi and figured out that poor sleep quality was in 67.2% of medical students with low physical activity vs. 25.7% with moderate or high physical activity. the study indicated that lost sleep because of late night computer use was very common among young men and women: almost every day – 11%, a few times per week – 15%, or a few times per month – 37% of participants. to determine the prevalence and clinicalpathological characteristics of upper fgid, records of questionnaires were evaluated. the most commonly presented complaints were heartburn, indigestion, epigastric pain/burning, excess throat mucous, or a lump in throat etc. these symptoms were absent in 30% of enrolled participants, but among the students who fulfilled the criteria for fgid, were more common in males (65.5%), than in females (34.5%). smoking causes a high risk for the prevalence of upper fgid. the data of the microscopic analysis of human saliva, namely samples of non-stimulated saliva microcrystallines, detects different types of distribution according to fgid (table 4). there were not any significant differences in saliva microcrystallines between the genders. the 1st first type of saliva microcrystallines was dominant among participants with symptoms of upper functional gid, because increased gastric acidity entailed the increase of saliva crystallization. both 2nd and 3rd crystallization types were the most common among the participants. it has been established that the 4th type of saliva microcrystallines is prevalent among medical students, who displayed both circadian disruption and physical inactivity. a growing amount of evidence argues that saliva could be a diagnostic tool for ans imbalance, which is represented by decrease of general adaptive reserve (20-22). microscopically, a large number of irregular crystal structures are seen in the 4th type of saliva crystallization (figure 1d, and figure 2e, f). investigation of stimulated saliva secretion didn’t show any significant differences in volumes and ph. the average ph of saliva was about 7.9, which is in the normal range, considered to be 5.6 to 7.9, and this keeps local balance in the oral cavity. figure 3 represents the crystal structure of the bio compounds in samples of saliva microcrystallines, which were determined using x-ray powder diffraction analysis according to standard procedure. at the beginning, the comparable characteristics of packet film and the dry precipitate of saliva samples with the 4th type of microcrystalline were tested in comparison for the elimination of wrong signals (figure 3a). phase analysis by x-ray diffraction confirmed that the microcrystalline samples were two-phase kcl (structural type nacl, with group fm–3m), parameter of unit cubic cell a = 6.3015(3) å, its volume v = 250.227 (18) å3 (figure 3b) with another compound with un-identified small reflex for 2θ = 27,78°. after standardization with theoretical diffractogram kcl (parameter of unit cubic cell a = 6.3060(4) å, value hkl for first reflex 200 (figure 3c) were a set of mild reflexes for the second phase (figure 3d). regarding several well-known data, parameter a for kcl could be from 6.26 to 6.3163 å; for k1-xnaxcl, x= 0-1, a=6.2916–5.6400 å. thus, the main bio compound in dry microcrystalline is pure kcl. the traces of sodium are very small (x ~ 0.01 in k1–xnaxcl). discussion this study confirmed that the intensive lifestyle of medical students related to their demanding educational process, modern behaviors of young people, and the lack of contractile duration of skeletal muscles, is quite common and could be a specific type of stress which causes imbalance in capacities for adaptation, leading to functional disorders in the very stresssensitive gastrointestinal tract. numerous data suggests impact dysfunction of brain-gut axis in induction fgid (23-26). regarding the final data, saliva represents a potential fluid for diagnostics of various diseases (13, 16, 17) and our results have shown its microcrystallines could be a diagnostic tool for stress-related gastrointestinal disorders. it is well known that epigenetic modifications play a crucial role in seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 65 southeastern european medical journal, vol 1, 2017. maladaptation and chronic stress-associated health outcomes. physiologically-based therapeutic implications of the student lifestyle should be included to recommendations of prevention and the treatment of fgid. in conclusion, the above observations underline the obtained results in saliva microcrystalline samples and suggest that an integrative view of saliva investigation could be novel very promising diagnostic tool for the early detection of changes in the human body. considering the obtained data about medical students’ modern lifestyle, the disturbance of daytime/nighttime rhythmic changes, and the prevalence of risk factors, we can conclude that these factors are involved in the changes in saliva microcrystalline formation. the obvious changes in saliva microcrystallines in persons with higher physical inactivity and circadian disruption may present external stimuli for the induction of upper functional gi disorders. a physiologically based program of increased duration of skeletal muscle contractility will be helpful in the prevention of functional digestive disorders and their health outcomes. acknowledgement the authors are grateful to pavlo demchenko and roman gladyshevskii from the department of inorganic chemistry of ivan franko national university of lviv, lviv, ukraine for performing xray diffraction methods on saliva samples. disclosure funding. no specific funding was received for this study. competing interests. none to declare. references 1. hamilton m. t., healy g. n., dunstan d. w., zderic t. w., owen n. too little exercise and too much sitting: inactivity physiology and the need for new recommendations on sedentary behavior. current cardiovascular risk reports. 2008;2(4):292-298. 2. karatsoreos i. n. effects of circadian disruption on mental and physical health. current neurology and neuroscience reports. 2012;12(2):218-225. 3. stevens rg, zhu y. electric light, particularly at night, disrupts human circadian rhythmicity: is that a problem? phil trans r soc b 2015 may 5;370(1667):20140120. 4. proper k. i., singh a. s., van mechelen w., chinapaw m. j. sedentary behaviors and health outcomes among adults: a systematic review of prospective studies. am j prev med. 2011;40(2): 174-182. 5. thorp a. a., owen n., neuhaus m., dunstan d. w. sedentary behaviors and subsequent health outcomes in adults: a systematic review of longitudinal studies, 1996–2011. am j prev med.. 2011;41(2):207-215. 6. haruma k., kinoshita y., sakamoto s., sanada k., hiroi s., miwa h. lifestyle factors and efficacy of lifestyle interventions in gastroesophageal reflux disease patients with functional dyspepsia: primary care perspectives from the legend study. internal medicine. 2015;54(7):695-701. 7. drossman da. the functional gastrointestinal disorders and the rome iii process. gastroenterology. 2006;130(5):1377-90. 8. zayachkivska o.s. the role of salivary endogenic bioregulators in the formation esophagoprotection at experimental injury of the esophagus. contemporary gastroenterology 2006;30 (4):65-71. 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(in russian). 17. lakhtin y., moskalenko p., levkov a., & smeyanov y. application of saliva crystalloscopy methods in the diagnosis of malignant tumours of salivary glands and sjögren disease (doctoral dissertation, published by iashe london), 2015. 18. wriedt m, näther c. synthesis, crystal structures, thermal and magnetic properties of new selenocyanato coordination polymers with pyrazine as co‐ligand. z anorg allg chem. 2011;637(6):666-71. 19. kraus w, nolze g. powder cell–a program for the representation and manipulation of crystal structures and calculation of the resulting x-ray powder patterns. j appl crystallogr. 1996;29(3):301-3. 20. petrakova l, doering bk, vits s, et al. psychosocial stress increases salivary alpha-amylase activity independently from plasma noradrenaline levels. schmidt u, ed. plos one. 2015;10(8):e0134561. doi:10.1371/journal.pone.0134561. 21. vineetha r, pai k-m, vengal m, gopalakrishna k, narayanakurup d. usefulness of salivary alpha amylase as a biomarker of chronic stress and stress related oral mucosal changes – a pilot study. j clin exp dent 2014;6(2):e132-e137. doi:10.4317/jced.51355. 22. tominaga, k., fujikawa, y., tsumoto, c., kadouchi, k., tanaka, f., kamata, n., arakawa, t. disorder of autonomic nervous system and its vulnerability to external stimulation in functional dyspepsia. j clin biochem nutr. 2016;58(2):161-5. doi: 10.3164/jcbn.15-140. 23. bonaz b. stress and the gastrointestinal system. in neuro-immunogastroenterology 2016 (pp. 123-156). springer international publishing. 24. pellissier s, bonaz b. chapter eleven-the place of stress and emotions in the irritable bowel syndrome. vitam horm 2017;103:32754. 25. brzozowski b, mazur-bialy a, pajdo r, kwiecien s, bilski j, zwolinska-wcislo m, mach t, brzozowski t. mechanisms by which stress affects the experimental and clinical inflammatory bowel disease (ibd): role of brain-gut axis. curr neuropharmacol 2016;14(8):892-900. 26. wiley jw, higgins ga, athey bd. stress and glucocorticoid receptor transcriptional programming in time and space: implications for the brain–gut axis. neurogastroenterol mot. 2016;28(1):12-25. seemedj 2022, vol 6, no 1 aspiration during vaccination 121 southeastern european medical journal, 2022; 6(1) review article aspiration during vaccination: evidence for sars-cov-2 vaccination 1 josip kajan 1*, marko sablić 2, marija heffer 1 1 department of medical biology and genetics, faculty of medicine, j. j. strossmayer university of osijek, osijek, croatia 2 department of anatomy and neuroscience, faculty of medicine, j. j. strossmayer university of osijek, osijek, croatia *corresponding author: josip kajan, jkajan@mefos.hr received: feb 25, 2022; revised version accepted: mar 22, 2022; published: apr 27, 2022 keywords: covid 19 vaccines, intravenous injection, vaccination abstract aspiration has always been performed during intramuscular vaccine injections to ensure that the needle does not puncture one of the blood vessels. however, at the beginning of the twenty-first century, this procedure became debatable. using an advanced search builder and logical operators, we searched the pubmed database for all articles about aspiration guidelines. the deltoid blood vessels are large and diverse, with potentially dangerous changes occurring in certain groups such as athletes or people with connective tissue diseases. the pharmacokinetics and reported side effects of improperly applied vaccines differ. some reported vaccine-related injuries, such as subacromial bursitis, can be avoided by using the aspiration technique. we discussed experiments that provide evidence that intravenous administration of mrna vaccines can cause myopericarditis. aspiration during vaccination is not technically demanding and does not require much time. previous arguments against aspiration were based on efforts to make the procedure of vaccinating children less painful. in response to public concern about vaccine-induced thrombotic thrombocytopenia as a possible side effect, denmark issued a guideline on mandatory aspiration during vaccination in march 2021. guidelines vary by country, and there is a need for an updated and globally applicable instruction manual. countries should carefully document vaccine side effects so that they could be compared between countries that aspirate and those who do not. more focused research experiments are needed to determine the relationship between aspiration and side effects. we propose a randomized study to compare the effectiveness of aspiration. (kajan j, sablić m, heffer m. aspiration during vaccination: evidence for sars-cov-2 vaccination. seemedj 2022; 6(1); 121-128) seemedj 2022, vol 6, no 1 aspiration during vaccination 122 southeastern european medical journal, 2022; 6(1) introduction the vaccination campaign against covid-19 focused public attention on the safety of vaccines and vaccine side effects. not only is the vaccine being called into question due to side effects, but so is the method of administering the vaccine. the aim of this article is to encourage reflection on vaccination techniques in the hope that a discussion on this issue will increase confidence in covid-19 vaccination and increase vaccination uptake. twenty years ago, aspiration was a common procedure during vaccination aspiration used to be a common procedure during intramuscular vaccine injections to ensure that the needle did not puncture any blood vessels. however, the guideline recommending this procedure was not evidence-based, and the procedure was disputed at the beginning of the twenty-first century (1). because of its unproven value, the who removed aspiration from intramuscular vaccine administration procedures in 2004 (2). according to plotkin and orenstein, aspiration was unnecessary because there were no large blood vessels at the site intended for intramuscular injections. in a more recent edition of their book, they cited the 2007 ipp study, which found the procedure to be painful, especially for infants (3,4). based on the who guidelines and the 5th edition of plotkin’s book (5,6), the official 2013 uk immunization guidelines, the green book, stated that aspiration was unnecessary. the centers for disease control and prevention guidelines confirmed this, citing an article that makes no mention of aspiration or the distance of large blood vessels from the injection site (7,8). similarly, the canadian government’s canadian immunization guide does not recommend aspiration (9). search strategy related to aspiration procedure using the and and or logical operators, we searched the pubmed database using the advanced search builder. the following search strategy was used: (((“intramuscular administration” [all fields]) or (“intramuscular injection” [all fields])) and ((“aspiration” [all fields]) or (“technique” [all fields]))) and ((“vaccine” [all fields]) or (“medication” [all fields])). table 1. search results with mentioning of aspiration s.no author publication year type of article sample size 1. nicoll and hesby(12) 2002 review 2. wynaden et al.(50) 2006 review and questionnaire 3. hunter(51) 2008 review 4. taddio et al.(52) 2009 review 5. mcdonnell et al.(53) 2010 research 6. ogston-tuck(54) 2014 review 7. taddio et al.(55) 2015 review 8. silva et al.(56) 2021 research 148* *randomized clinical trial that compared adverse events with or without aspiration seemedj 2022, vol 6, no 1 aspiration during vaccination 123 southeastern european medical journal, 2022; 6(1) there were 77 results in total, with 8 articles mentioning aspiration and 17 articles discussing medication and vaccine administration but not mentioning aspiration (see table 1). we also searched the reference and citation lists of the studies that were included. aspiration as part of intramuscular injection was first mentioned in john h. stokes’ 1945 book modern clinical syphilology: diagnosis, treatment, case studies (10). although the book was not available to us, we found a figure from it in a 1961 article by zelman, which simply stated the importance of aspiration (11). the most detailed instructions for needle aspiration were provided by nicoll and hesby in 2002: after inserting the needle into the muscle, the first step is to pull back the plunger for 5-10 seconds; if blood is aspirated into the syringe, the administration should be stopped and the needle withdrawn from the muscle; the syringe with blood should be discarded; new syringe should be prepared and administered at a new administration site (12). the blood vessels in the muscle are large and diverse the deltoid muscle is a common vaccination site. it is well supplied with blood from several sources: the deltoid and acromial branches of the thoracoacromial artery, anterior and posterior branches of the humeral circumflex artery and deltoid branches of the deep brachial artery. these arteries are accompanied by large veins that drain into the axillary and cephalic veins (13,14). the intersection of the anteroposterior axillary line and the perpendicular line from the mid-acromion is the safest area for intramuscular injections. the lower half of the muscle is avoided due to the significant risk of injury to the arteries and the axillary nerve (15), whereas the upper third is avoided due to the risk of hitting the subacromial bursa. complications involving the subacromial bursa have been reported after unintentional injection of covid-19 vaccines into the bursa (16). the deltoid’s excellent blood perfusion alters the pharmacokinetics of the injected medication and causes absorption at this site to be faster than at other intramuscular injection sites (17). furthermore, athletes have a thinner arterial wall and a larger vessel diameter, which might make them more susceptible to aneurysms and ruptures (18,19). the importance of blood supply to muscles is also demonstrated by the avoidance of intramuscular administration in people with hemophilia due to frequent formation of intramuscular hematomas (20). renewed interest in vaccine administration during covid-19 pandemic the ongoing worldwide vaccination campaign against covid-19 has reignited the interest in vaccine administration (21). newspaper articles, letters to the editor, and scientific discussions on youtube channels have all addressed the topic of aspiration (22–26). we searched the pubmed database for articles published between 2019 and the end of 2021 that contained one of the following terms: vaccine administration method, vaccine administration methods, vaccine administration practices, vaccine administration recommendations, vaccine administration technique, vaccine administration techniques. there were fifteen studies found, four of which discussed vaccine administration and one of which discussed covid-19 vaccine administration (27–30). pharmacokinetics of mrna vaccine during intravenous and intramuscular administration during intramuscular administration, the peak concentration of any drug is lower and delayed, and it is higher and instantaneous during intravenous administration (31). to induce immunity, mrna vaccines must be translated into spike protein (32). there have been no studies that compare the pharmacokinetics of mrna vaccines administered intramuscularly versus intravenously, but an in vivo study examined the pharmacokinetic translation of mrna luciferase delivered by lipid seemedj 2022, vol 6, no 1 aspiration during vaccination 124 southeastern european medical journal, 2022; 6(1) nanoparticles (33). intravenous administration led to a shorter translation half-life and higher total protein formation (33). because solid lipid nanoparticles, carriers for the mrna of the spike protein, have different affinities for various tissues due to their charge (34), they should be carefully monitored in trials. for bnt162b2 vaccine, the distribution of lipid nanoparticles has been reported only for the liver, spleen, adrenal glands and ovaries, while the rest of the data is not publicly available (35). all of these differences raise the question of whether they are significant enough to affect immunogenicity and thus create more potent side effects. intravenous injection of mrna vaccine may induce myopericarditis in a mouse model an increased incidence of myocarditis and pericarditis was observed following mrnabased covid-19 vaccination (36). the link between myopericarditis and vaccination has yet to be established. li et al. compared intramuscular and intravenous vaccine administration in a mouse model with saline as a control. despite the fact that the intramuscular group exhibited significant weight loss and higher serum cytokine concentrations after the first dose, only the intravenous group developed a histopathological picture of myopericarditis. histological changes in the intravenous group lasted for two weeks and worsened significantly after the second dose, regardless of whether the injection was intramuscular or intravenous. according to the authors, these in vivo findings show that intravenous administration of mrna vaccines can cause myopericarditis (37). there is also evidence that spike protein can cause pericarditis on its own via cd147 receptor signaling (38). intravenous injection of adenovirus causes thrombocytopenia in a mouse model adenovirus vectors are another type of covid19 vaccine vectors. adenovirus-induced thrombocytopenia was first observed some twenty years ago, with the introduction of gene therapy (39,40). vaccine-induced thrombotic thrombocytopenia has also been observed as a side effect of adenovirus-based covid-19 vaccines (41). these vectors were first effective in in vivo gene delivery transduction, and their advantage is that most cells have adenovirus receptor on their membrane, making adenovirus vectors easily infectable (42). gene therapy requires high adenovirus concentration via intravenous injection because of the quantitative transduction of the entire organ (40). this high viral particle concentration in the blood may cause adenovirus-induced thrombocytopenia via the following mechanism: the virus attaches to and activates platelets via the coxsackie adenovirus receptor. after activation, platelets expose p-selectins on their membranes and aggregate with leukocytes. platelet and leukocyte aggregates then activate other leukocytes and endothelial cells. this type of abnormal platelets is phagocytosed by macrophages. macrophages and activated endothelial cells release large amounts of highmolecular-weight forms of von willebrand factor and microparticles. as a result, von willebrand factor and microparticles cause a strong pro-coagulant stimulus (39). there is also a more recently proposed mechanism of action that is similar to heparin-induced thrombocytopenia and is caused by the presence of anti-platelet factor 4 (43). the mechanism of vaccine-induced immune thrombotic thrombocytopenia is initiated by the complex of adenovirus vector and platelet factor 4. this complex induces lymphocytes b cells, which produce anti-platelet factor 4. this type of antibody stimulates platelets, monocytes and neutrophils. activated platelets and monocytes release pro-coagulant microparticles, while activated neutrophils exert a pro-inflammatory effect (44). this complex interaction between adenovirus vector chadox1 and platelet factor 4 was confirmed by baker et al in 2021 (45). at the end of march 2021, denmark issued a guideline for mandatory seemedj 2022, vol 6, no 1 aspiration during vaccination 125 southeastern european medical journal, 2022; 6(1) aspiration during intramuscular vaccine administration following the increased public concern about vaccine-induced thrombotic thrombocytopenia, denmark issued a guideline on mandatory aspiration during vaccination. the english version of the guideline can only be found in the janssen vaccination procedure instructions on the statens serum institute website (46). a population-based cohort study from denmark that followed participants from 1 october 2020 to 5 october 2021 found 69 cases of myocarditis or myopericarditis within 28 days of vaccination. data from this study does not correspond to the data from united states and israel indicating an increased rate of myocarditis and myopericarditis in men aged 12-39 (47). on 17 february 2022, the robert koch institute published an update for covid-19 vaccination, stating that vaccines should only be administered intramuscularly and that aspiration during vaccination makes sense for increased safety to avoid intravascular injection (48). conclusion aspiration during vaccination is neither technically demanding nor time-consuming. arguments against it were based on efforts to alleviate a painful procedure when vaccinating children, thereby reducing stress and achieving better child cooperation. although one study examined the safety of the injection site on the deltoid muscle, its sample size was insufficient and unrepresentative (49). larger sample size studies are required to support or refute the safety of aspiration omission during vaccination. there are two study types that could be used. the first type should be designed to determine the prevalence of positive blood aspirations during intramuscular injections. the second type should compare the number of side effects associated with intramuscular injections with and without aspiration. the previous study, which found that intravenous injection of mrna vaccine caused myocarditis in mice, failed to establish a cause-and-effect relationship, but did indicate the potential presence of a preventable factor. furthermore, the results of that experiment cannot be translated to humans. all countries should document vaccine side effects much more thoroughly, allowing comparisons to be made between countries where aspiration is performed and those where it is not. although findings obtained in this manner would not be sufficient to demonstrate a causal relationship between side effects and aspiration, they would provide sufficient evidence to begin a more direct research experiment. acknowledgement. the authors thank jelka petrak, mark cooper and ana marusic for suggestions and assistance. disclosure funding. this article received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. competing interests. none to declare. references 1. sepah y, samad l, altaf a, halim ms, rajagopalan n, javed khan a. aspiration in injections: should we continue or abandon the practice? f1000research. 2014; 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revised version accepted: april 26, 2017; published: may 9. 2017 keywords: pharmacogenomics, medicine, clinical, pediatrics influence the drug response of an individual patient (2). since inter-individual variabilities in drug response are often genetically determined, the field of pharmacogenetics evolved for the purpose of assessing the influence of specific genetic biomarkers on the efficacy and safety of drugs. pharmacogenomics, which emerged from pharmacogenetics due to the appearance of genome-wide association studies (gwas), analyses the entire genome to find multigenetic factors related to an individual’s drug response, abstract pharmacogenomics is a promising field which increasingly influences medicine and biomedical research in many areas. the aim of this article is to review recent advancements in the understanding of genetic polymorphisms and their influence on interindividual variability in drug response. also, the main variabilities in drug response according to sex differences will be discussed. the translation of pharmacogenomics into the clinical routine as well as the challenges of achieving the goal of personalized medicine are also discussed. the role of pharmacogenetic tests in pediatrics has not been well defined yet, but it is clear that those tests could help in resolving some issues regarding the administration of drugs to children. at the conclusion, the foremost ethical, social and regulatory issues regarding the translation of pharmacogenomics into clinical practice and future perspectives in the field will be discussed. (bozic i, omanovic t, kuna l, kizivat t, smolic r, vcev a, smolic m. pharmacogenomics: sex differences and application in pediatrics. seemedj 2017;1(1);108-120) seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 109 southeastern european medical journal, vol 1, 2017. representing another step towards personalized medicine (2-4). furthermore, pharmacogenomics represents a promising area for the aims of maximizing the benefits of pharmacotherapeutic regimens and minimizing the risks of developing adrs, which are significant causes of morbidity and mortality in patients worldwide (5-7). most genetic biomarkers that have been found to affect the drug response in pharmacogenetic and pharmacogenomic studies are variations in the dna sequences of genes encoding both enzymes and transporters included in the absorption, distribution, metabolism and excretion of drugs (8). pharmacogenetic and pharmacogenomic trials are usually designed by three different methodologies: candidate gene studies, genome-wide association studies, as well as whole-exome and whole-genome sequencing (9). in candidate gene studies, a hypothesisdriven approach is used, where a single targeted gene, encoding a protein included in the metabolism of the drug, is investigated (9, 10). however, in gwas, two groups of patients with a different drug response profile are compared, and potential associations with many known genetic variants are investigated (9). the third and most comprehensive methodology is whole-exome and whole-genome sequencing, where all human genetic material is analyzed for variants related to drug efficacy and safety. however, the analysis of data collected in such studies remains difficult (9). genetic polymorphisms of drugmetabolizing enzymes so far, data obtained from previous pharmacogenomic studies has yielded extensive reports about the genetic influence on treatment outcomes and side effect appearance (11). polymorphisms in genes encoding drugmetabolizing enzymes (dmes), or transporters in phases i and ii of the drug metabolism, often influence the drug response and determine the risk for the development of adrs (12). single nucleotide polymorphisms (snps), minor insertions or deletions, as well as the amplification and deletion of gene copies, are genetic polymorphisms of dmes (12). more than 90% of human genes contain at least one snp. consequently, so far over 14 million snps have been discovered in the human genome (13). factors that cause variations in drug response are complex and involve fundamental aspects of human biology (1). however, several variants of dmes have been discovered since the completion of the human genome project (1). as a reward for this laborious work, genetic polymorphism studies show clinically significant applications (1). altogether 57 cytochrome (cyp) genes encode cyp enzymes, which are subdivided into families, mainly involved in the metabolism of exogenous substances (14). since cyp450 takes part in the metabolism of 65-70% of clinically used drugs, those enzymes play an especially significant role in the field of pharmacogenomics. in fact, cyp450 enzymes are responsible for 75-80% of all reactions of the phase i metabolism (15,16). for instance, 20-25% of all marketed drugs are metabolized by cyp2d6, which is why its polymorphisms are very frequently investigated in pharmacogenomic trials (17,18). for example, it is the rate-limiting enzyme in the catalyzing process of tamoxifen’s conversion intoactive metabolites, 4-hydroxytamoxifen and endoxifen. therefore, cyp2d6 has a significant impact on the individual’s response to tamoxifen therapy; as well it does for many other drugs (19). a further example of the numerous cyp450 enzymes under pharmacogenomic investigation is cyp2c19, which catalyzes reactions in the metabolism of clinically important drugs, such as tolbutamide, glipizide, phenytoin, warfarin and flurbiprofen. more than 20 polymorphisms of genes encoding this enzyme have been studied, whereas the two most common variant alleles, cyp2c19*2 and cyp2c19*3, are null alleles, exhibiting reduced enzymatic activities (1, 18). seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 110 southeastern european medical journal, vol 1, 2017. in addition, gwas data confirmed previous discoveries, indicating the importance of dme variants in drug response (for instance, clopidogrel and cyp2c19), thus offering evidence for different levels of correspondence (12). also, more than 50% of the drugs used in clinical practice are metabolized by cyp3a4 in the human liver. so far, more than 20 variants of cyp3a4 are known. the frequency of individual cyp3a4 variants varies greatly across different ethnic groups and many of those variants exhibit altered enzyme activities, which means that polymorphisms of the cyp3a4 gene could be responsible for the difference in drug response between ethnic groups (1, 18, 20). recent studies have also emphasized the critical role of cyp2c19 polymorphisms for the therapeutic effects of clopidogrel. furthermore, the importance of cyp2d6 polymorphisms for tamoxifen treatment appears to be relevant (12). importantly, non-cyp450 dmes also play important roles in the metabolism of various drugs. therefore, polymorphisms of genes encoding those dmes also influence the therapeutic effect and could lead to potential adverse reactions after drug intake (1). pharmacokinetics and pharmacodynamics: sex difference even though the mechanisms are still not well understood, it seems that sex also plays a crucial role in the pharmacological response (21)(22). generally, it is known that pharmacokinetic differences between men and women are more extensive compared with variations in pharmacodynamics (21). some drugs frequently prescribed for the treatment of cardiovascular diseases, such as verapamil and amlodipine, show sexual dimorphism in their pharmacokinetic profile (14). bioavailability of amlodipine slightly differed among sexes, with women showing higher bioavailability. however, after the adjustment of the obtained data to body weight, no significant sgd in the bioavailability of amlodipine remained (23). some recent findings suggest that sex differences appear also in the field of pharmacogenomics (21). studies about sex-based differences in the response to pharmacotherapy show that women experience adrs in a more frequent manner, and that those side effects may also be more severe compared with the adrs in men (thiazolidinedione-induced bone fractures, iatrogenic long qt syndrome and iatrogenic systemic lupus erythematosus, etc.) (23). for instance, inhibitors of angiotensin converting enzymes (aceis) and antagonists of angiotensin receptors 1 (arbs) are important compounds of therapeutic regimens in the treatment of cardiovascular diseases (14). investigations have reported that during treatment with aceis, cough and angioedema are more frequent in women than in men. furthermore, it has been shown that the xpnpep2 c-2399a genotype is associated with an increased frequency of aceiassociated angioedema in black men, but not in white men and women (21, 22). because of this circumstantial evidence for the unfavorable safety profile in women, further studies should be conducted to clarify sex-gender related differences (23). importantly, sex differences in drug response could have various reasons, and they are not all caused by the varieties of the dna sequence of pharmacogeneticallyrelevant genes (24). due to the fact that not many genes that are known to be relevant for pharmagenomic studies are located on sex chromosomes, sexgender differences in drug response are possibly caused by differences in autosomes, transcriptional gene regulation due to sexspecific epigenetic modifications, posttranscriptional modifications or by the effect of sex hormones (24, 25). although animal studies suggest that sex-specific expression of cyp isoforms is a common case in rodents, this mechanism is subtler in humans (20). however, sex dimorphic metabolism does occur also in humans, for instance in the case of cyp3a4, as a very important isoform of cyp, which is expressed more extensively in females than in males (20). seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 111 southeastern european medical journal, vol 1, 2017. furthermore, evidence for sex-gender dependent gene expression exists, and this sexually dimorphic expression is seen in liver, muscle, fat and brain tissue (26, 27). also, frequencies of specific allelic polymorphisms are unequally distributed among men and women, which also contributes to differences in the drug response and safety profile (24). furthermore, psychological, physiological and lifestyle factors influence sex-gender differences, in particular in the case of therapy outcomes for drugs targeting the central nervous system (28,29). it is still unknown whether pathological conditions influence pharmacokinetic and pharmacodynamic parameters in a sex-gender dependent way (28, 29). until now, sex differences have been undervalued, and the study design in clinical trials during the drug approval process did not have a proper sex-based approach, all of which has led to a deficient understanding of drug response and side effect disposition among women, a deficiency that is a missing link in the path towards personalized medicine (21, 25). it is of great importance to carry out further studies with a more sex-based approach, so that it would become possible in the future to better adjust pharmacotherapeutic regimens and individual drug doses according to the sex of the patient (21). translating pharmacogenomic knowledge into clinical practice the main long-term goal of pharmacogenomics is to translate observations regarding the genetic basis of drug responses into a more effective and less toxic treatment for individual patients in the everyday clinical practice (30). until now, due to detailed pharmacogenomic studies for some drugs, the clinical application of this branch of personalized medicine has already become possible (9). examples of successful clinical application of pharmacogenomics are listed in table 1 (9). pharmacogenetic testing, in order to determine the suitability of a drug for the individual patient, is gradually moving from specialty medications, for instance drugs prescribed for patients with cancer, to more broadly prescribed medications, as are statins, codeine or warfarin (31). heretofore, the drug labels of 137 medications include pharmacogenetic-related information (32). it has been estimated that 16 percent of medications prescribed in primary care are pharmacogenetically impacted (33). the delivery modes of pharmacogenomic testing are yet unclear; therefore, the main goal is to write clinical practice guidelines with decisionmaking algorithms informed by controlledclinical pharmacogenomics trials (34-37). these guidelines may increase precision, accuracy and the relevance of recommendations and subsequent applicability (38). although pharmacogenomics offers significant potential to improve the clinical outcome of individual patients, the translation of pharmacogenomic knowledge and principles into clinical practice has been slow in most settings (30). one of the reasons could be the fact that in many cases, the variability of drug response involves many different factors other than pharmacogenomics (1). in fact, the understanding of the pathophysiological processes of the disease, the identification of important genes, as well as the recognition of the roles the genetic polymorphisms of receptors, transporters or dmes play in the pharmacological outcomes, are all required for the complex process of achieving individualized medicine (1). for the reason that many factors that are not reflected in genomic information influence drug toxicity and efficacy, it is questionable whether personalized drug treatment will ever become attainable by pharmacogenetic testing alone (1). in addition to scientific difficulties, economic, ethical, social and regulatory issues are also very challenging (1). pharmacogenomic applications in pediatrics during the past decade, much effort has been devoted to improve the safety and efficacy of medical products used for the treatment of the pediatric age spectrum, from premature newborns to adolescents, with a special accent seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 112 southeastern european medical journal, vol 1, 2017. on the progress in dosing strategies. since the medicine registration protocols of the european union, before the legislation called paediatric regulation in 2006, did not obligate pharmaceutical companies to assure in advance a pediatric investigation plan (pip), as reported by the european commission, between 50% and 90% of the drugs used in pediatric medical care were administered off-label, without being adequately tested nor authorized with a tolerable risk benefit profile in children (39-41). there are two main issues regarding the administration of drugs to children under these circumstances. on the one hand, doses for children are usually obtained empirically in the absence of evidence by adapting adult doses to body weight in a trial-and-error pattern (42). since absorption, disposition, metabolism and drug elimination are subject to developmental processes due to the ontogeny of dmes, pharmacokinetic responses are not equivalent to those during adulthood (40, 43-45). thus, dosing protocols for children should be independently obtained in pediatric studies. on the other hand, along with changes in enzymatic activity due to the changes of gene expression during the development process, which is specific for the pediatric population, the influence of snps of genes for some receptors, enzymes and transporters involved in drug response and metabolism, also plays a fundamental role in the accurate prediction of treatment response in children (44, 46). table 1. examples of clinically relevant pharmacogenomic testing. drug genetic variants influence notes warfarin anticoagulant drug cyp2c9 gene and vkorc1 (78) genetic polymorphisms impact dose requirements of warfarin therapy (9, 79) only 59% of us patients have inr 2-3 (because of the narrow therapeutic range of warfarin many patients still receive an incorrect dose) (80) simvastatin lipid-altering agent used to treat hypercholesterolemia sl-co1b1*5 (81) increased risk of statininduced myopathy patients suffering from cardiovascular disease (82) simvastatin is a widely used drug: sl-co1b1 testing could reduce the incidence of statininduced myopathies or rhabdomyolysis(83) codeine opioid analgesic drug cyp2d6 (84) genetic polymorphisms affects metabolizing phenotype of patients (84) metabolizing phenotypes: ultra rapid (high risk for adrs) and extensive metabolizers to intermediate and poor metabolizers (inadequate analgesia) (84) geftinib egfr tyrosine kinase inhibitor (tki) egfr (85) egfr mutation is a biomarker of geftinib efficacy (86, 87) brca1 as well as compounds of the nf-κb pathway also affect the response of egfr mutated patients to geftinib (88) irinotecan chemotherapeutic drug used for colorectal cancer ugt1a1*28 (89) ugt1a1*28 polymorphism leads to lower protein expression and irinotecan-induced toxicity (hematological and digestive adrs) (89, 90) irinotecan is a prodrug of sn-38 that is conjugated via ugt1a1 (90) seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 113 southeastern european medical journal, vol 1, 2017. consequently, additional pharmacogenomic trials should be performed in children if relevant biomarkers are available, since only about 70% of pharmacogenomic data obtained from studies on adult populations can be applied to children (45, 47-50). the role of pharmacogenomic tests in pediatrics has not been well defined yet and there is a lack of genotype-guided dosing strategies for children (51-53). compared to adults, children have an increased level of complexness due to physiological maturation processes, as well as the ontogeny of gene expression, which contributes to the influence of specific genetic variants investigated in pharmacogenomic trials (54). as a result, the approach to results from pharmacogenomic trials on children should differ substantially from those yielded in studies on adult patients (54). even though the dna sequence persists throughout life, the pattern of gene expression is dynamic, and changes in protein synthesis occur. indeed, some of the most important enzymes involved in drug metabolism, like cyp450 and udp glucuronyl transferase, display gene expression depending on developmental changes (55). for instance, cyp3a7 gene expression is already observable in the fetal liver during the first trimester of pregnancy, but the level of cyp3a7 enzyme production decreases after the immediate postnatal period. as the cyp3a7 level of gene expression decreases, cyp3a4 and cyp3a5 expression boost after the end of the first postnatal week, until they reach 30% of the adult enzyme activity levels by the end of neonatal period (55, 56). although the overall level of cyp3a protein expression remains constant, differences in specificities to substrates, as well as metabolic and catalytic capacities exist, depending on whether the prevailingly expressed genetic variant is cyp3a7 or cyp3a4 (55). thus, drug metabolism via the cyp3a subfamily varies due to developmentally regulated gene expression and, depending on the moment of drug administration during the neonatal period, genetic variants in different members of the cyp3a subfamily are important. for instance, this might play an important role in altering the pharmacokinetics of tacrolimus, a calcineurin inhibitor and immunosuppressant drug used in children and adults after solid organ transplantation (57, 58). since tacrolimus has a very narrow therapeutic index, it is still challenging to adjust the dosing regimen in children so that further pharmacogenomic trials and its implementation into clinical practice could potentially be crucial in this process (43, 59). due to the breakthrough of pharmacogenomics in the field of personalized medicine, a number of pharmacogenomic studies are being conducted, mainly with adults as subjects in those trials. however, even though pharmacogenomic research on children lags behind, results published so far in this field accentuate the difference between children and adults in the framework of pharmacogenomics (54, 60). therefore, it would be vague to directly extrapolate data from adult pharmacogenetic trials without putting them into the variable pharmacokinetic context of a developing child (54, 61). indeed, there are some well-known clinical utilizations of pharmacogenetic tests in adult medical care, but there is still a lack of knowledge to translate those results to the pediatric patient spectrum (60). the most significant achievements in this study field have been made in pediatric hematology and oncology, but some trials were also conducted in the areas of rheumatology, endocrinology, neurology, gastroenterology, pulmology and organ transplantation in children (46, 54). for example, one of the most frequent chronic diseases in children is juvenile idiopathic arthritis (jia), which can lead to persistent disabilities in adulthood (65). methotrexate (mtx) is a diseasemodifying antirheumatic drug (dmard) important in the treatment of jia that unfortunately exhibits heterogeneity in the clinical response (64). pharmacogenomic studies suggest that polymorphisms in many genes encoding products included in the disposition and biochemical pathways of mtx, for instance, cacna1i, zmiz1, tgif1 or cftr, can affect the clinical outcome and therefore have the potential to make pharmacogenetic testing of individual patients an integral diagnostic component before the application of mtx therapy (62-64). seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 114 southeastern european medical journal, vol 1, 2017. moreover, since pharmacogenomics seems to be one of the emerging tools used to improve drug safety by the avoidance of giving specific drugs to susceptible individuals who are likely to develop adverse drug reactions (adrs), it is important to consider that some adrs are specific to, or more frequent in, children. in fact, it is in some cases unreasonable to assume the genetic influence on the occurrence of some adrs through adult pharmacogenomics study results, if these adrs are unique to the child population (54). ethical issues concerning pharmacogenomics apart from the recognized benefits of pharmacogenomics in the future perspective of patient medical care, considerable ethical and legal questions, which could eventually overwhelm the unprepared legal framework, are arising (66). first of all, pharmacogenetic testing should not be administered without a signed informed consent, containing appropriate information about the benefits and risks of the procedure (67). due to a child’s inability to fully understand, or understand at all, the purpose and possible aftereffects of pharmacogenetic testing, parents or legal guardians must sign the consents. the ethical issue addressed here is that a person who signs the informed consent is not the person who receives the genetic testing, which could have unforeseeable consequences (68). as well as in other genetic tests, the dna sample primarily used for one purpose, in this case, obtaining data about the individual efficiency and safety of a patient's treatment could unintentionally yield additional secondary information. secondary information could be information about a predisposition to several diseases, a prognosis of current illness, or pharmacogenetic information about drugs used for other conditions, which could lead to both a patient’s psychological and economical discomfort. for example, if the patient with a disease would be classified as a nonresponder to drugs currently used for the treatment of his condition, this could have an impact on the patient’s insurance payments or even lead to discrimination in their search for employment because of the additional health care costs for employers (68, 69). consequently, this example also raises the question of confidentiality and who should have access to data obtained by these tests. also, if the patient, for instance, undergoes testing for the apolipoprotein e genotype, which is the most frequently investigated pharmacogenetic biomarker for statins, he might also receive unwanted information about his risk of developing alzheimer’s disease in his elderly years (70, 71). also, due to the possible high costs of pharmacogenetic testing, it is likely that the economic status, whether of an individual patient or of a whole country, will influence and limit the accessibility of the method. correspondingly, this could enhance the unethical socioeconomic divisions and inequalities in the health care system (72, 73).the pathway towards personalized medicine entails that the drug development by pharmaceutical companies should be genetically guided. due to the further costs involved in such drug design, pharmaceutical companies might first want to evaluate the frequencies of alleles of interest. in the case of limited profits from the restricted drug market due to rare alleles and genotypes of nonresponders to available drugs, pharmaceutical companies might not be intrinsically stimulated to develop new drugs for those individuals. just like in the case of orphan drugs for rare diseases, drug development for rare, orphan genotypes should be economically stimulated (72, 74, 75). in addition to the problem of orphan genotypes, it could be the case that developing drugs even for very frequent genotypes would not be of interest to major companies if the genotypes were geographically located in socioeconomically poorer areas (72). despite the ethical and legal questions, it seems that pharmacogenetic testing is one of the most promising steps towards personalized medicine. however, effort should be put into establishing legal parameters that can cope with the emerging needs of the evolving field of pharmacogenomics. seemedj 2017, vol 1, no. 1 implementation of the n terminal prob-type natriuretic peptide test… 115 southeastern european medical journal, vol 1, 2017. future perspectives further trials with even more participants are likely to yield results in the near future that could extend the number of clinical implementations and make another step towards personalized medicine (76). since the field of pediatric pharmacogenetics still falls behind the research on adults, advances in the research field are still expected so that the complex compound of genetic influence and ontogenic dynamics in children could be understood. a better and more profound explanation of those processes would certainly facilitate the clinical implementation of the future collected knowledge in the field of children's pharmacogenomics (51). furthermore, it is of great importance to educate clinicians about data interpretation of pharmacogenetic test results so that they can gain the required knowledge to accurately stratify patients into high-risk or low-risk groups regarding drug toxicity and consequently improve the therapeutic outcome without putting susceptible patients at risk of predictable life threatening adrs. therefore, new user-friendly and up-to-date guidelines should be provided to clinicians in order to help the future implementation of pharmacogenomic study results into the clinical daily routine (43, 46, 77). it is likely that the further use of next-generation sequencing will lead to new advances in pharmacogenetics (43). also, although still expensive, high-throughput screening methods could become more affordable in the future and help progress in the scientific field (77). hence, these and further technological improvements could upgrade the current knowledge in pharmacogenomics to an advanced level, which could lead to more clinical aims, consequently increasing the safety of drugs used for the treatment of many diseases. however, since there are also emerging ethical concerns, an adequate legal framework should be established. conclusion pharmacogenomics is a rapidly emerging and promising scientific field in which an increasing amount of studies are being conducted. although there are still challenges, it is promising that they could vanish with the improvement of study designs and the formation of international cooperation that would validate pharmacogenomic study results and promote the clinical use of pharmacogenetic tests (76). sex-related differences have been reported in pharmacogenomics trials. since more severe and more frequent drug adverse reactions have been found in women, whose pharmacological status is less studied, emphasis should be put on pharmacogenomic investigation in women (34). there are still no satisfactory data present regarding pediatric pharmacogenomic studies (54). however, legislations, both in the eu and usa, accentuate the need for clinical trials on pediatric patients so that an admissible level of safety in drug administration could be reached (40). the process of achieving individualized medicine for many diseases is complex, especially considering that many nongenetic factors influence drug toxicity and drug efficacy. in addition to scientific difficulties, economic, ethical, social and regulatory issues are also very challenging (1). acknowledgements the support of the j. j. strossmayer university of osijek research grants (to ms and to av) are gratefully acknowledged. disclosures competing interests: none to declare. 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thalassotherapia opatija, specialized hospital for medical rehabilitation of cardiac, pulmonary and rheumatic diseases, opatija, croatia 4 university of rijeka, faculty of health studies, rijeka, croatia *corresponding author: lovorka bilajac, lovorka.bilajac@medri.uniri.hr received: mar 1, 2022; revised version accepted: apr 7, 2022; published: apr 27, 2022 keywords: older people, falls, functional status, physical activity abstract aim: falling can have serious consequences for older persons, but fear of falling is also a current problem. the aim of the study is to assess the incidence of falls and fear of falling among older adults, as well as to study the connection of falls and fear of falling with functional ability, body mass index (bmi) and age. methods: the participants of this study are older persons ( > 60 years) who live in the city of rijeka, are mobile and have no symptoms of dementia. the relationship between falls, fear of falling, body mass index (bmi) and functional ability was measured using various indicators. assessment of physical functioning and pain relief was made using the coop/wonca questionnaire. falls and fear of falling were assessed using questions with different response options. results: the results of the study showed that participants who reported experiencing fear of falling (fof) had a higher bmi (p = 0.018) and did not feel physically healthy (70%). participants who lived with a partner reported experiencing fof at a lower rate (36.4%). the most frequently reported functional problems were visual problems (46.2%) and walking difficulties (40.0%). conclusions: a large percentage of older persons struggle with the effects of aging, which include various health problems that can increase the risk of falling and fof. prescription of physical activity and engaging in it could improve functional ability and have an important effect on healthy aging. this could be the starting point from which key stakeholders can plan different types of public health interventions, prescribing physical activity to maintain functional ability that enables well-being in old age. (bilajac l, šulavjak d, zulle k, vasiljev v, juraga d, marinović glavić m, rukavina t. physical activity as prediction of functional ability among elderly. seemedj 2022; 6(1); 83-91) seemedj 2022, vol 6, no 1 physical activity and elderly 84 southeastern european medical journal, 2022; 6(1) introduction the aging process brings with it many physiological changes that can subsequently lead to the development of risk factors and chronic diseases. according to the world health organization, most changes begin after the age of 60 and usually include functional impairment (such as hearing loss, visual impairment, reduced mobility) and the onset of noncommunicable diseases (heart disease, stroke, chronic lung disease, malignancies and dementia) (1). all these conditions lead to functional impairment and consequently cause dependence. falls are a major cause of morbidity and mortality in older persons. it is estimated that 30–40% of persons over the age of 65 fall at least once a year (2). the number of fall-related deaths in persons over 75 years of age in the us increased from 8613 to 25189 from 2000 to 2016, which means that the number of older persons who died due to falling nearly tripled in 2016. several studies in europe report a similar increase in the number of deaths among older persons due to falling (3, 4). despite this, most falls in the general population do not endanger the individual’s life, but 37.3 million falls per year require some form of medical intervention (5) and 70% of these result in some degree of disability (6). falling can lead to moderate to severe musculoskeletal injuries, loss of independence and even death. however, one of the most important psychological consequences of falls is the appearance of fear of falling (fof) (2). fof leads to physical, functional, psychological and social changes in older persons, which are associated with a decrease in physical activity and health. falls could also be a consequence of fof. according to a 2013 study, persons who have fallen in the past year are usually over 85 years old, have cataracts, significant circulatory, respiratory, musculoskeletal and nervous system disorders, take four or more different medications, use walking aids and are overweight (6). obesity is associated with a 25% higher risk of falling than in persons of normal weight. this information is explained by the fact that being overweight affects the postural stability of older persons, which can lead to more frequent falls (7). despite all these facts, there is a simple way to prevent most of these conditions. physical activity has significant health benefits for hearts, bodies and minds. regular physical activity provides significant benefits for health and can improve muscular and cardiorespiratory fitness, improve bone and functional health, reduce the risk of hypertension, coronary heart disease, stroke, diabetes, various types of cancer (including breast cancer and colon cancer) and depression, reduce the risk of falling as well as hip or vertebral fractures and help maintain a healthy body weight (1, 8). the aim of this article was to show the functional ability of elderly persons who engage in physical activity, as well as to study and establish a relationship between risk factors that influence the possibility of falling and the prevalence of fear of falling in the elderly as a function of functional ability. materials and methods the survey is retrograde and cross-sectional and takes into account the first measurements of functional status of the elderly from 2016. the sample includes respondents/elderly persons who participated in group exercise sessions which were organized as part of the urban health centres europe project and were maintained as continuation of the project. the group exercise sessions were organized twice a week and lasted for one hour. the exercises were led by physiotherapy students who were trained to lead exercise sessions for the elderly. older persons also participated in educational programs for active and healthy aging. participants the participants in this study are older persons (over 60 years old) who live in the city of rijeka, are mobile, live independently and have no dementia symptoms. recruitment was organized during project activities, and visiting nurses recruited participants based on the seemedj 2022, vol 6, no 1 physical activity and elderly 85 southeastern european medical journal, 2022; 6(1) inclusion criteria. all participants were informed about the study and signed an informed consent form in order to participate in the study. confidentiality was maintained in the storage, retrieval and analysis of the data. the study was approved by the ethics committee for biomedical research of the faculty of medicine, university of rijeka. methods the dartmouth functional health assessment charts (coop/wonca questionnaire) were used to assess physical functioning and pain. the coop questionnaire is used for a quick subjective assessment of the subjects’ functional health. the coop charts reflect the patient’s assessment of their functional capacity (9). the instrument consists of six charts. for this study, we used physical functioning and pain charts. respondents rated the intensity of physical activity they had been capable of in the last two weeks on a scale of 1 to 5, where 1 is very heavy activity and 5 is very light activity. in addition, participants rated the intensity of pain in the last four weeks on a scale of 1 to 5, where 1 means that there was no pain and 5 means that the pain was severe. participants’ height and weight were measured at baseline and bmi was calculated as weight (in kilograms) divided by the square of height (in meters) (kg/m2). in order to detect falls in the past 12 months, respondents were asked “have you fallen in the last 12 months?”, where the answer that could be given was yes or no. if the answer was yes, another question was asked, “how many falls did you have in the last 12 months?”. fof was measured by asking the question “in the last months, did you worry about falling down?” the responses were yes or no, and we classified it as a binary variable. measurements of restrictions on daily activities and data on the use of medical devices such as walking sticks, glasses etc. were collected. sociodemographic data information on age group, gender, education, marital status and living arrangements were collected. results were analyzed in terms of experience of falls, demographic characteristics (age, gender and marital status) and personal perceptions of health and functional ability. statistical analysis statistical analysis included a descriptive analysis of participants’ baseline characteristics. pearson’s chi-squared test and fisher’s exact test were used to compare the characteristics of those with and those without fear of falling, as reported. these tests were used to determine the level of statistical significance at the 0.05 (5%) level. for p ≤ 0.05, the difference in arithmetic mean is considered significant. data were processed using statistica (version 13.5.0.17, 1984–2018 tibco software inc) and microsoft office excel 2016. results sixty-five older persons participated in the study, of whom 93.9% (n = 61) were women and 6.1% (n = 4) were men. the mean age of the participants was 69.8 (± 7.3) years, with the youngest participant being 60 years old and the oldest 92 years old. subjects were divided into three age groups: younger than 65, 66 to 70 and over 70 years old. the average bmi of the subjects was 27.5 (± 3.5) kg/m2; for men, it was 28.8 (± 4.7) kg/m2 and for women, it was 26.6 (± 3.3) kg/m2. half of the subjects were classified as pre-obese. most of the respondents live with a partner (table 1). of the health problems observed in the lives of the respondents, visual impairment (46.2%) and walking difficulties (40.0%) were the most frequently reported. in addition, almost 40.0% of respondents reported problems in everyday life due to physical fatigue. problems with balance (15.4%) and hearing (12.3%) were mentioned less frequently (table 1). seemedj 2022, vol 6, no 1 physical activity and elderly 86 southeastern european medical journal, 2022; 6(1) table 1. characteristics of the study population groups overall male female n 65 (100.0) 4 (6.1) 61 (93.9) average age 69.83 (± 7.3) 81 (± 7.3) 69.17 (± 6.7) age group younger than 65 20 (30.8) 20 (32.8) 65 to 70 18 (27.7) 18 (29.5) older than 70 27 (41.5) 4 (100.0) 23 (37.7) bmi (kg/m2)* 27.5 (± 3.5) 28.8 (± 4.7) 26.6 (± 3.3) normal weight 22 (34.4) 1 (25.0) 21 (35.0) pre-obesity 32 (50.0) 2 (50.0) 30 (50.0) obesity class i 10 (15.6) 1 (25.0) 9 (15.0) marriage status* unmarried married 34 (53.1) 3 (75.0) 31 (51.7) extramarital union 1 (1.6) 1 (1.7) divorced 9 (14.1) 9 (15.0) widow/widower 20 (31.2) 1 (25.0) 19 (31.6) household composition** living alone 22 (34.9) 1 (25.0) 21 (35.6) living with a partner, no children 26 (41.3) 2 (50.0) 24 (40.6) living with a partner and children 6 (9.5) 1 (25.0) 5 (8.5) living without partners and children 6 (9.5) 6 (10.2) living in a household that i share with others 3 (4.8) 3 (5.1) problems in everyday life resulting from walking difficulties 26 (40.0) 3 (75.0) 23 (37.7) balance problems 10 (15.4) 2 (50.0) 8 (13.1) impaired hearing 8 (12.3) 1 (25.0) 7 (11.5) impaired vision 30 (46.2) 1 (25.0) 29 (47.5) hand weakness 23 (35.4) 3 (75.0) 20 (32.8) physical fatigue 25 (38.5) 3 (75.0) 22 (36.1) * one subject did not respond ** two subjects did not respond . fof is present in half of the respondents and increases with age. for example, more than 60.0% of respondents over 70 years of age reported fof, while fof was present in 25.0% of respondents under 65 years of age. less than a quarter of the respondents reported experiencing a fall in the past year. there was no difference in the presence of fof between the respondents who had experienced a fall in the past 12 months and those who had not (p < 0.05). the results of the study showed that the respondents who reported experiencing fof had a statistically significantly higher bmi than those who did not report experiencing this fear (p = 0.018). despite the higher bmi among subjects who reported experiencing a fall in the past 12 months compared to those who did not, this difference was not statistically significant. subjects who were married reported experiencing fof at a significantly lower rate (36.4%) than those who were unmarried (63.3%). seemedj 2022, vol 6, no 1 physical activity and elderly 87 southeastern european medical journal, 2022; 6(1) they also reported falling at a lower rate (20.6%) compared to the unmarried respondents (26.7%). however, no statistically significant difference was found between the subjects in that regard (table 2). table 2. comparison of functional ability, falls and fear of falling in the study group fear of falling* fall in the last 12 months present n (%) not present n (%) p present n (%) not present n (%) p participants 32 (50.0) 32 (50.0) 15 (23.1) 50 (76.9) male 3 (75.0) 1 (25.0) 1 (25.0) 3 (75.0) female 29 (48.3) 31 (51.7) 14 (23.0) 47 (77.0) age group younger than 65 5 (25.0) 15 (75.0) 5 (25.0) 15 (75.0) 65 to 70 10 (58.8) 7 (41.2) 2 (11.1) 16 (88.9) older than 70 17 (63.0) 10 (37.0) 8 (29.6) 19 (70.4) bmi (kg/m2) 27.7 (± 3.6) 25.6 (± 3.1) < 0.05 27.2 (± 3.7) 26.6 (± 3.4) marriage status* married 12 (36.4) 21 (63.6) < 0.05 7 (20.6) 27 (79.4) other 19 (63.3) 11 (36.7) 8 (26.7) 22 (73.3) perception of physical health n (%) feel physically healthy 25 (46.3) 29 (53.7) 14 (25.5) 41 (74.5) do not feel physically healthy 7 (70.0) 3 (30.0) 1 (10.0) 9 (90.0) pain in everyday life n (%) do not report pain or report mild pain 17 (39.5) 26 (60.5) < 0.05 7 (15.9) 37 (84.1) < 0.05 moderate to severe pain 14 (70.0) 6 (30.0) 8 (40.0) 12 (60.0) problems in everyday life resulting from: n (%) walking difficulties present 17 (65.4) 9 (34.6) < 0.05 5 (19.2) 21 (80.8) not present 15 (39.5) 23 (60.5) 10 (25.6) 29 (74.4) balance problems present 9 (90.0) 1 (10.0) < 0.05 3 (30.0) 7 (70.0) not present 23 (42.6) 31 (57.4) 12 (21.8) 43 (78.2) impaired hearing present 5 (62.5) 3 (37.5) 3 (37.5) 5 (52.5) not present 27 (49.1) 29 (52.7) 12 (21.1) 45 (78.9) impaired vision present 20 (66.7) 10 (33.3) < 0.05 8 (26.7) 22 (73.3) not present 12 (35.5) 22 (64.7) 7 (20.0) 28 (80.0) * one subject did not respond subjects who felt physically unwell reported experiencing fof at a higher percentage (70.0%) compared to subjects who felt physically healthy (46.3%). fof occurs significantly more often in subjects reporting moderate to severe pain in everyday life (70.0%) compared to subjects reporting no pain or mild pain. in addition, subjects reporting moderate to severe pain reported a significantly higher percentage of falls (40.0%) compared to subjects reporting seemedj 2022, vol 6, no 1 physical activity and elderly 88 southeastern european medical journal, 2022; 6(1) no pain of this intensity in everyday life (15.9%). subjects reported a significantly higher percentage of falls when they had problems with walking (65.4%), balance (90.0%) and vision (66.7%) in everyday life compared to subjects who reported no such problems (table 2). discussion according to the results of our study, the presence of fof is associated with several factors. elderly persons with fof have higher bmi, are not married, live alone, have moderate to severe pain and functional problems in everyday life, have a negative perception of their physical health and are of advanced age. the risk factors for fof occurrence are interrelated and interdependent. studies have shown that the annual incidence of falls in older persons is between 30 and 40% (2), but it was less than 25% in our study population. we can assume that the reason for this could be the participation in group exercise sessions twice a week. the association between female gender and more frequent falls is frequently mentioned in the literature (10, 11). in our study, the small number of male subjects must be taken into account, along with their higher average age compared to the female subjects. a higher incidence of falls is also associated with older age (12). a psychological consequence of the fall could be the appearance of fof, and on the other hand, fof could be the reason for the fall. falls occurred significantly more often in those subjects who suffer from moderate to severe pain in everyday life. older persons who report pain in everyday life are more likely to report experiencing a fall in the past 12 months than those who report no pain. the results of our study show that respondents with moderate to severe pain reported a higher percentage of falls in the past 12 months compared to those who did not report such pain. the study associated an increase in the incidence of falls with pain affecting activities of everyday life (13). a 2018 study states that pain is one of the most important factors in the occurrence of moderate to severe mobility impairment in older persons (14). elderly women who live alone are more likely to suffer from fof, which is associated with impaired quality of life and reduced functionality in performing activities of everyday life. we can say that less than 40% of older persons limit their activities due to fof. fof can occur before a person experiences a fall (12). non-married respondents have a significantly higher incidence of fof than married respondents; they also report a higher percentage of falls. this is consistent with the findings of a 2010 study, which found that persons who live alone are at higher risk of falling than persons who live with family (15). walking is one of the basic activities of everyday life. the presence of gait instability can be a significant risk factor for the occurrence of falls, especially in older persons (16, 17). the results of our study show that persons who reported problems with gait difficulty were less likely to have fallen in the past 12 months than persons who reported no such difficulties. we can assume that they are more careful in their everyday movements, but the results show that fof occurs in more than 65% of respondents who reported walking difficulty. the findings can be explained by a 2017 study which found that postural instability and gait problems associated with fof are linked to a higher incidence of falls in older persons. research has shown that the occurrence of fof only predicts future falls in persons with postural instability and gait problems (18). in 2014, hoang et al. associated fof with previously experienced falls, poor balance and negative perceptions of physical health (19). in our study, results also showed that fof was more common in subjects who had previously experienced a fall. the subjective feeling of balance problems in everyday life is more pronounced in persons who have a fear of falling. according to our research, 90% of persons who reported balance problems have fof. the results suggest that persons who have a negative perception of their physical health are more likely to report experiencing fof than persons who have a positive perception of their health, which is related to the study by hoang et seemedj 2022, vol 6, no 1 physical activity and elderly 89 southeastern european medical journal, 2022; 6(1) al. (19). fof was present in 70% of subjects with a negative perception of health. fof and its frequency are associated with visual and hearing impairments (20, 21), especially when a person has both, together with balance problems (22). in addition, persons who perceive their hearing impairment as a significant disability are at higher risk of experiencing two or more falls within five years than persons who do not have hearing problems (21). participants who reported having hearing problems reported a higher percentage of falls in the past 12 months. choi and ko published a paper in 2015, in which they found that persons with vision problems fall more often than persons who do not have such problems (12). the reason for this could be a reduced perception of environmental signs. in addition, subjects who perceived problems related to visual impairment reported a higher percentage of fof when performing functional activities of everyday life. studies show that visual impairment is an independent risk factor for the occurrence of falls, their recurrence and the occurrence of fractures (20, 21, 23, 24). obesity is associated with an increased risk of falls, as well as a higher risk of disability in activities of everyday life following falls in older adults (25). in addition to the incidence of falls and disability, obesity is associated with a greater likelihood of pain and reduced mobility after falls when compared to peers with normal body weight (26). a 2011 study found no significant difference between fall experiences in individuals with normal weight and overweight individuals (27), in contrast to a 2017 study, which found that undernourished and obese individuals had a higher percentage of falls compared to individuals with normal weight and overweight individuals (28). older persons who have postural instability and are obese are at higher risk of falling because obesity affects balance and performance of dynamic tasks (29). some studies state that older women who are obese have less postural stability than those with normal body weight, which increases the fear of falling (30). the results of our study showed that subjects who reported experiencing a fall in the past 12 months had, on average, a higher bmi than those who did not report experiencing a fall. furthermore, fof increased with increasing bmi, meaning that subjects who reported experiencing a fall had a higher bmi on average. research suggests that obese persons are at increased risk of falling, but that such falling does not necessarily lead to serious injury. in such cases, obesity becomes a protective factor against musculoskeletal injuries (6, 25, 27, 30). a disadvantage of this study is the small number of subjects and the disproportionate number of male and female subjects, as well as the fact that the severity of falls, the number of falls and the severity of fof were not considered. fof is higher in subjects reporting problems in everyday life related to walking difficulty, balance problems, vision problems, bmi and moderate to severe pain, which is consistent with the published literature. the falls themselves are more likely to occur in persons who report moderate to severe pain. physical activity can improve balance and thus prevent the possibility of falls and increase the selfconfidence of the elderly and reduce the incidence of fear. the findings and data from other studies suggest that a large percentage of older persons struggle with the effects of aging, which includes various comorbidities and health problems that can increase the risk of falling and fof. the results of the research showed that both previous falls and fear of falling are important predictors of functional ability. they equally impact the development of everyday activity limitations. physical activity can help the elderly stay physically independent. acknowledgement. none. disclosure funding. no specific funding was received for this study competing interests. none to declare. seemedj 2022, vol 6, no 1 physical activity and elderly 90 southeastern european medical journal, 2022; 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67(5):475-81. doi: 10.6061/clinics/2012(05)12. author contribution. acquisition of data: lb, dš, kz, vv, dj, mmg, tr administrative, technical or logistic support: lb, dš, kz, vv, dj, mmg, tr analysis and interpretation of data: lb, dš, kz, vv, dj, mmg, tr conception and design: lb, dš, kz, vv, dj, mmg, tr critical revision of the article for important intellectual content: lb, dš, kz, vv, dj, mmg, tr drafting of the article: lb, dš, kz, vv, dj, mmg, tr final approval of the article: lb, dš, kz, vv, dj, mmg, tr guarantor of the study: bn, km, gi, jj, pv, ja, ks, bjn , jb provision of study materials and patients: kz, dj, mmg, tr statistical expertise: dj, mmg, tr seemedj 2022, vol 6, no 1 hypertension management with sglt2i 1 southeastern european medical journal, 2022; 6(1) invited review changing the landscape of hypertension management with sglt2i 1 ines bilić ćurčić 1,2, vjera ninčević 3, silvija canecki varžić 2,4, ivana prpić križevac 2,4, jasminka milas ahić 2,5, ivica mihaljević 6,7 1 department of pharmacology, faculty of medicine, josip juraj strossmayer university of osijek, croatia 2 clinic for internal medicine, university hospital center osijek, croatia 3 department of pharmacology and biochemistry, faculty of dental medicine and health, josip juraj strossmayer university of osijek, croatia 4 department of internal medicine, faculty of medicine, josip juraj strossmayer university of osijek, croatia 5 department of pathophysiology, faculty of medicine, josip juraj strossmayer university of osijek, croatia 6 clinical institute of nuclear medicine and radiation protection, university hospital center osijek, croatia 7 department of nuclear medicine and oncology, faculty of medicine, josip juraj strossmayer university of osijek, croatia *corresponding author: ines bilić ćurčić, ibcurcic@gmail.com received: mar 23, 2022; revised version accepted: apr 4, 2022; published: apr 27, 2022 keywords: sglt2i, blood pressure, type 2 diabetes, cardiovascular disease abstract sodium-glucose cotransporter-2 inhibitors (sglt2i) are a newer class of drugs that have primarily been used in the treatment of type 2 diabetes. however, as new findings from clinical trials have become available, their indication has been expanded to include treatment of heart failure and chronic kidney disease without the presence of diabetes. the pathophysiological mechanisms of extraglycemic effects of sglt2i are still being unraveled, but one of the most prominent consequences is a decrease in blood pressure, which has implications for hemodynamics and arterial stiffness. recent findings indicate that this class of drugs has a beneficial effect on lowering nocturnal blood pressure (bp), with special importance in type 2 diabetes (dmt2), since unregulated nocturnal hypertension is associated with an increased incidence of cardiovascular (cv) events. in this minireview, we have summarized current knowledge about the effects of sglt2i on blood pressure, including office, home, and ambulatory bp, and potential implications for treatment of hypertension in diabetic and non-diabetic individuals, with positive effects on cardiorenal outcomes. (bilić ćurčić i, ninčević v, canecki varžić s, prpić križevac i, milas ahić j, mihaljević i. changing the landscape of hypertension management with sglt2i. seemedj 2022; 6(1); 1-11) seemedj 2022, vol 6, no 1 hypertension management with sglt2i 2 southeastern european medical journal, 2022; 6(1) introduction sglt2i are a class of newer therapeutic agents in diabetes treatment with proven cardiorenal benefits, which reduce the risk of cardiac failure and death from cardiovascular disease (cvd) and slow the progression of diabetic kidney disease [1–5]. this class of agents is therefore recommended for diabetes treatment in highrisk patients with heart failure, cvd or kidney disease, based on recent guidelines [6]. in addition, their role in the treatment of heart failure and kidney disease independent of the presence of diabetes has also been recognized recently [7, 8]. possible mechanisms responsible for cardiorenal benefits are numerous, including positive effects on hemodynamic parameters, reduction of arterial stiffness, improvement of chronic inflammation, metabolic fuel switching, along with traditional mechanisms, such as lowering of blood glucose and reduction of blood pressure and body weight [9–12]. diabetic patients have a specific 24-hour bp profile with a non-dipper pattern of nighttime bp likely caused by increased circulating volume [13–16]. the presence of nocturnal hypertension is more common in patients with diabetes compared to those without it [17–19] and is associated with higher mortality rates [20]. likewise, the frequency of masked hypertension is relatively high in diabetic patients, ranging from 27% to 47% [21–24], and it is one of the independent predictors of cvd [24]. recognizing and treating masked hypertension in patients with dmt2 is imperative, and out-of-office bp measurement using home bp monitoring (hbpm) or ambulatory bp monitoring (abpm) is therefore recommended in the diagnosis of hypertension (htn) [25–27]. effects of sglt2i on bp measurements sglt2i agents, such as empagliflozin [28–34], canagliflozin [35–39], ertugliflozin [40–42] and dapagliflozin [43–47], have been shown to lower office blood pressure in patients with dmt2 and htn in a number of studies. according to several published meta-analyses, marked reductions in systolic bp (sbp) ranged from -2.45 mmhg to 4.45 mmhg compared to active comparators, with diastolic bp (dbp) reductions from -1.46 mmhg to -2.01 mmhg [48, 49]. changes in bp with empagliflozin and existing antihypertensive therapy were examined in the sacra (sodiumglucose cotransporter-2 [sglt2] inhibitor and angiotensin receptor blocker [arb] combination therapy in patients with diabetes and uncontrolled nocturnal hypertension) study. the study included japanese patients with type 2 diabetes and poorly controlled nocturnal hypertension who were receiving standard antihypertensive therapy and were randomized to the empagliflozin or the placebo group. clinic bp, 24-hour abpm and morning home bp was monitored, while the primary endpoint was change from baseline in nighttime bp (abpm). at 12 weeks, empagliflozin significantly reduced daytime, 24-hour, morning home, and clinic sbp (-9.5, -7.7, -7.5, and -8.6 mmhg, respectively). body weight and glycosylated hemoglobin reductions between groups were significant, albeit minor (-1.3 kg and -0.33 percent, respectively). in addition, marked reductions in n-terminal pro-b-type natriuretic peptide levels were observed in the empagliflozin versus the placebo group ( 12.1%; p = 0.013); reductions were likewise observed in atrial natriuretic peptide levels ( 9.7%; p = 0.019) [50]. however, there was no statistically significant reduction in overnight bp compared to the placebo group, with a drop in nighttime sbp of 6.3 mmhg from the baseline. given that a 5-mmhg decrease in mean overnight sbp has been linked to a 20% reduction in cvd risk [51], the nighttime bp reduction observed with empagliflozin could have clinical importance regardless of the lack of statistical significance. as previously mentioned, 24-hour ambulatory bp is a better predictor of cv risk than office bp [20, 24]. a recently published meta-analysis including randomized, double-blind, placebocontrolled trials reporting 24-hour abpm data demonstrated the lowering effect of sglt2i on ambulatory systolic and diastolic bp by 3.76 mmhg (95 percent ci, 4.23 to 2.34; i2 = 0.99) and 1.83 mmhg (95 percent ci, 2.35 to 1.31; i2 = 0.76), seemedj 2022, vol 6, no 1 hypertension management with sglt2i 3 southeastern european medical journal, 2022; 6(1) respectively, over a 24-hour period. there were also significant reductions in systolic and diastolic bp during the day and at night, independent of body weight change [52]. another meta-analysis examining the effects of sglt2i inhibition on ambulatory bp aimed to assess the relationship between dose and ambulatory bp response to sglt2 inhibition and to compare it to low-dose hydrochlorothiazide. according to this meta-analysis, in 24-hour abpm, sglt2 inhibitors caused an average reduction of 3.62/1.70 mmhg in systolic/diastolic bp, which is equivalent to the bp-lowering efficacy of low-dose hydrochlorothiazide, regardless of the sglt2 inhibitor dose. however, sglt2 inhibition reduced blood pressure more effectively during the day than at night [53]. in a post-hoc analysis of the empa-reg bp trial, empagliflozin was found to lower nocturnal bp more than daytime bp in patients with dmt2 and a non-dipper htn profile [54]. figure 1. pathophysiological mechanisms involved in blood pressure-lowering effects of sglt2i and their role in preventing cv and kidney disease the presently published data regarding the effects of sglt2i on home bp are limited to the japanese population. in addition to the results of the previously mentioned sacra study, which showed a lowering effect of empagliflozin on morning home bp [50], the results of the shiftj study demonstrated a beneficial effect of canagliflozin on nocturnal, morning and evening home sbp (-5.23, -6.82, -8.74, respectively) compared to the control group of patients with uncontrolled dmt2 and nocturnal bp [55]. in addition, beneficial effects of dapagliflozin on morning, evening and nocturnal home sbp (8.32; -9.57 and -2.38 ± 7.82 mmhg, respectively) seemedj 2022, vol 6, no 1 hypertension management with sglt2i 4 southeastern european medical journal, 2022; 6(1) were reported in japanese patients with dmt2 [56]. based on the available evidence, we can conclude that the drop in 24-hour ambulatory bp seen with sglt2 inhibitors is a class effect and is comparable to low-dose hydrochlorothiazide. likewise, it seems that sglt2i have a significant bp-lowering effect at night, suggesting that these drugs have the potential to repair altered circadian bp rhythms in hypertensive patients with dmt2, thus improving cardiovascular and kidney outcomes (figure 1). proposed mechanisms of sglt2 inhibition on blood pressure the effect of sglt2i on lowering blood pressure could be explained by several mechanisms involving changes in hemodynamics due to a decrease in effective circulating volume caused by diuresis and natriuresis, reduction in body weight and uric acid levels, antihyperglycemic effects, shifting metabolic fuel from glucose to ketones, inhibition of sympathetic nervous system activity, change in arterial stiffness and switching of salt-sensitive to non-salt sensitive bp phenotype due to osmotic diuresis [11, 57, 58] (figure 1). microvascular and macrovascular dysfunction leading to increased arterial stiffness is common in diabetic patients [59, 60], meaning that an increase in blood pressure and blood pressure variability cannot be attenuated in these patients’ major arteries and are instead conveyed to atherosclerotic plaque sites in the periphery. as a result, the combination of htn and dmt2 creates hemodynamic stress that predisposes patients to the development of cvd [61–63]. according to preclinical evidence, improvements in arterial stiffness and endothelial function may contribute to the cv advantages of sglt2i therapy [64]. this is supported by evidence obtained in clinical trials, suggesting that currently available sglt2i medications reduce arterial stiffness in individuals with dmt2 [65–68]. furthermore, dapagliflozin also reduced cardiac fibrosis in infarcted rat hearts by controlling macrophage polarization via stat3 signaling, as well as by altering epicardial fat tissue distribution and cytokine and adipokine secretion [69–71]. clinical evidence of beneficial effects of sglt2i on cardiorenal outcomes large cardiovascular outcome trials (cvot) have shown the beneficial effects of sglt2i on improving cv outcomes, largely due to reduction in hospitalizations caused by heart failure and, in some studies, decreasing mortality. in the empa-reg outcome trial, empagliflozin significantly reduced the occurrence of primary endpoint events (3-point major cardiovascular event (mace) consisting of non-fatal myocardial infarction, non-fatal stroke and cv death) compared to placebo, with significantly lower rates of cv death, hospitalization for heart failure (hf) and death from any cause [1]. similar results were obtained in the canvas trial [2], which examined the cardiovascular safety of canagliflozin, demonstrating a reduction in 3-point mace, but no effect on cv death, as opposed to the empareg outcome trial. dapagliflozin, on the other hand, reduced the risk of hf, but had no effect on atherosclerotic cvd, as shown in the declare-timi 58 trial [3]. according to available evidence, the protective effect of sglt2i on 3-point mace is limited to patients with previously existing cvd, while class benefits exist for hf regardless of cvd presence [72]. furthermore, in patients with dmt2 and chronic kidney disease (ckd) (egfr 60 ml/min/1.73 m2), a favorable effect on hf and 3-point mace has been confirmed [73]. recently, results from two large-scale studies were published, demonstrating improved outcomes in patients with hf with reduced ejection fraction (hfref) treated with dapagliflozin and empagliflozin (dapa-hf and emperor-reduced, respectively) independent of the presence of diabetes [7, 8]. based on the evidence obtained in the above trials, the guidelines of the european society of cardiology (esc) propose sglt2i and/or seemedj 2022, vol 6, no 1 hypertension management with sglt2i 5 southeastern european medical journal, 2022; 6(1) glucagon-like peptide-1 receptor agonists (glp1 ra) as the treatment of choice for patients with diabetes and proven cvd or risk thereof, which is contrary to the common practice of using metformin as the first choice for dmt2 treatment [6]. nearly all patients included in the credence study, which examined the effect of canagliflozin on renal outcomes, were treated with angiotensin-converting enzyme (ace) inhibitors or angiotensin receptor blockers (arbs), with egfr from 30 to 90 ml per minute [74]. the experiment was halted early due to favorable interim analysis results, showing that the relative risk of composite renal outcomes was reduced by 34%, while the relative risk of end-stage renal disease was reduced by 32%. these findings reveal that renoprotection was accomplished across the whole range of egfr levels, re-establishing the nephroprotective effect regardless of baseline renal function. the probability of dialysis, transplantation, or death due to kidney disease (hard renal outcomes) was considerably reduced in individuals treated with sglt2i, as was recently validated in a meta-analysis of all studies using sglt2i in diabetic patients [72]. because of their reduced antihyperglycemic efficacy, sglt2i were previously not recommended for diabetic patients with egfr of less than 45 ml/min per 1.73 m2 [75, 76]. such restrictions became debatable after the evidence of renoprotective effects emerged in the mentioned trials [77]. this was confirmed in the latest clinical trials involving diabetic and non-diabetic patients with heart failure and preserved ejection fraction [7, 8], demonstrating improvement of hard renal outcomes in the lower specter of egfr, 30 and 20 ml/min/1.73 m2 in the emperor-reduced and dapa-hf trials, respectively. the final proof that the renoprotective effect is independent of the antihyperglycemic effect was provided by the dapa-ckd study, which indicated that dapagliflozin reduced renal events in patients with ckd with or without dm treated with maximum tolerated doses of ace inhibitor/arb [78]. beneficial effects were observed on noncv and all-cause mortality. conclusions the effects of sglt2i on blood pressure also appear to be largely responsible for the beneficial effects of this class of drugs on cardiorenal outcomes. they play a particularly important role in the control of nocturnal blood pressure, which is associated with increased cv risk, especially in patients with dmt2. thus, the use of sglt2i in patients with dmt2 and hypertension is certainly justified regardless of glycemic control in order to reduce the risk of cv and renal complications and improve the patient’s quality of life. the extent to which the effects of sglt2 inhibition on blood pressure are responsible for reduction of risk of heart failure, cardiovascular and renal disease in non-diabetic individuals with arterial hypertension remains a question to be answered. acknowledgement. none. disclosure funding. this work was supported by vif 2022 ip5. competing interests. none to declare. references 1. zinman b, wanner c, lachin jm, fitchett d, bluhmki e, hantel s, mattheus m, devins t, johansen oe, woerle hj, broedl uc, inzucchi se; 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